JP2023526551A - Liquid formulation of long-acting conjugates of glucagon derivatives - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a liquid formulation of a long-acting conjugate of a glucagon derivative and a method for producing the same.

Description

The present invention relates to liquid formulations of long-acting conjugates of glucagon derivatives and methods for producing the same.

Due to recent economic development and changes in eating habits, the incidence of metabolic syndrome-related diseases, including various diseases such as obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, diabetes, and liver disease, is rapidly increasing. is. Although such diseases may occur individually, in most cases they are closely related to each other and occur with various symptoms.

In particular, according to the World Health Organization (WHO), more than 1 billion adults are overweight worldwide, of which at least 3 million are clinically obese, with an estimated 250,000 annually in Europe. 000, and more than 2.5 million deaths worldwide each year are associated with overweight.

Obesity is a serious disease that causes various diseases as a worldwide disease, but there is a tendency to believe that obesity can be overcome by individual self-help efforts. However, obesity is surprisingly not easy to treat because it is a complex disease associated with mechanisms of action in appetite regulation and energy metabolism. Therefore, in order to treat obesity, not only the patient's own efforts but also methods for treating abnormal mechanisms of action related to appetite regulation and energy metabolism must be performed at the same time. Efforts are continuing to develop drugs that can treat pneumothorax.

As a result of the above efforts, anti-obesity agents such as Rimonabant (Sanofi-Aventis), Sibutramine (Abbott), Contrave (Takeda), Orlistat (Roche) have been developed, but they are fatal. However, it has the disadvantages of showing serious side effects and being inadequately effective in treating obesity. As such, researches are being actively conducted to develop new medicines that can solve the problems of conventional anti-obesity drugs, and glucagon derivatives have recently been the focus of attention.

Glucagon is produced in the pancreas when blood sugar begins to fall due to medications or other causes such as disease, hormone or enzyme deficiencies. Glucagon signals the liver to break down glycogen and release glucose, thus raising blood sugar levels to normal levels. Glucagon has also been reported to be effective in treating hypoglycemia. The hypoglycemic therapeutic effects of glucagon include stimulating the breakdown of hepatic glycogen into glucose (glycogenolysis) and increasing glucose production (glucose biosynthesis) derived from amino acid precursors leading to increased glucose efflux from the liver. This is the result.

In addition, glucagon has been reported to have an anti-obesity effect by suppressing appetite and activating hormone-sensitive lipase in adipocytes to promote lipolysis, in addition to raising blood sugar. However, glucagon has been limited in its use as a therapeutic agent due to its low solubility and precipitation at neutral pH.

Therefore, glucagon with improved physical properties may help treat severe hypoglycemia and increase lipolysis and β-oxidation in the liver. ) is useful for the treatment of

Drugs for treating hypoglycemia include diazoxide, octreotide, glucagon and the like. Of these, glucagon is currently used as a frozen dosage form due to its low solubility and precipitation at neutral pH. Its use has been limited due to frequent dosing when used as a hyperinsulinism therapeutic agent.

Under such a technical background, the present inventors have developed a derivative in which the amino acid sequence of glucagon is partially modified in order to improve the physical properties of glucagon and enhance its therapeutic effect on hypoglycemia and obesity (International Published Patent WO2016/108586 and International Published Patent WO2017/003191). It was confirmed through in vitro activity measurement that the developed glucagon derivative activates the glucagon receptor. Also, to develop long-acting conjugates of said glucagon derivatives with increased in vivo half-life, said long-acting conjugates exhibiting improved solubility and high stability at neutral pH due to altered pi different from native glucagon. was confirmed (International Publication WO2017/003191).

International Publication WO2016/108586 International Publication WO2017/003191 International Patent Publication No. WO97/34631 International Patent Publication No. 96/32478 International Publication WO2007/021129

H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979

There remains a need for the development of stable liquid formulations that allow long-term storage of long-acting conjugates of glucagon derivatives without fear of viral contamination.

One object of the present invention is to provide liquid formulations of long-acting conjugates of glucagon derivatives.

Another object of the present invention is to provide a method for producing the liquid preparation.

The liquid formulation according to the present invention has the advantage of providing storage stability to the conjugate of the present invention, which has a large molecular weight, in a simple dosage form, and can be economically provided.

This is the result of confirming the stability of long-acting glucagon derivative conjugates depending on the type of buffer substance. Specifically, compositions shown in Table 3 of Example 2 (compositions #1, #3, and #5 in Table 3) were used as liquid preparations of long-acting conjugates of glucagon derivatives. Stability results are shown after 7 weeks of storage at °C. Among the results, sodium acetate and histidine preparations as buffer substances showed the highest stability at 25°C for 7 weeks.

One aspect embodying the invention is a liquid formulation of a long-acting conjugate of a glucagon derivative. The long-acting conjugate refers to a substance in which a peptide having activity against a glucagon derivative is covalently linked to an immunoglobulin Fc segment via a linker.

In one embodiment, the present invention comprises a pharmacologically effective amount of a long-acting conjugate of a glucagon derivative peptide, wherein the glucagon derivative peptide and an immunoglobulin Fc segment are linked together, i) a buffer substance and ii) a sugar alcohol. liquid formulations of long-acting conjugates of glucagon derivative peptides comprising non-albumin-containing stabilizing agents comprising, sugars or combinations thereof. The long-acting conjugate refers to a substance in which a glucagon derivative peptide is covalently bound to an immunoglobulin Fc segment.

As one specific example, the liquid formulation is characterized by being a long-acting conjugate liquid formulation comprising a long-acting conjugate represented by the following chemical formula (1); a buffering substance; and a sugar alcohol, sugar, or a combination thereof. do:

X-La-F

In the chemical formula (1), X is a glucagon derivative peptide,
L is a linker,
a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
F is the immunoglobulin Fc segment;
- indicates a covalent bond:

[General formula 1]
Y-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T- X30 (general formula 1, SEQ ID NO: 46)

In the general formula 1,
X2 is α-methyl-glutamic acid, Aib (aminoisobutylic acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S) or D-serine;
X7 is threonine (T), valine (V) or cysteine (C);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X13 is tyrosine (Y) or cysteine (C);
X14 is leucine (L) or cysteine (C);
X15 is aspartic acid (D), glutamic acid (E) or cysteine (C);
X16 is glutamic acid (E), aspartic acid (D), serine (S), α-methylglutamic acid, or cysteine (C), or absent;
X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C), or valine (V), or absent ;
X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), or cysteine (C), or absent;
X19 is alanine (A), arginine (R), serine (S), valine (V), cysteine (C), or absent;
X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), α-methylglutamic acid, or cysteine (C), or absent ;
X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine (C), or absent;
X23 is isoleucine (I), valine (V), arginine (R), or absent;
X24 is valine (V), arginine (R), alanine (A), cysteine (C), glutamic acid (E), lysine (K), glutamine (Q), α-methyl-glutamic acid, or leucine (L) or does not exist;
X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or absent;
X28 is glutamine (Q), lysine (K), asparagine (N), arginine (R), or absent;
X30 is cysteine (C) or may be absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

The Aib means aminoisobutyric acid. As used herein, "Aib" may be mixed with "2-aminoisobutyric acid" or "aminoisobutyric acid".

A liquid formulation according to any one of the foregoing embodiments, wherein in said glucagon derivative peptide long-acting conjugate, said glucagon derivative peptide comprises an amino acid sequence of general formula 2:

Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-NT- X30 (general formula 2, SEQ ID NO: 47)

In the general formula 2,
X7 is threonine (T), valine (V) or cysteine (C);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X15 is aspartic acid (D) or cysteine (C);
X16 is glutamic acid (E) or serine (S);
X17 is lysine (K) or arginine (R);
X20 is glutamine (Q) or lysine (K);
X21 is aspartic acid (D) or glutamic acid (E);
X24 is valine (V) or glutamine (Q);
X30 is cysteine (C) or absent (except when the amino acid sequence of general formula 2 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

A liquid formulation according to any one of the foregoing embodiments, wherein said liquid formulation comprises 18-936 nmol/mL of said long-acting conjugate of formula (1); pH of said liquid formulation is 4.8-6.5; and 1.0-20% (w/v) of a sugar alcohol, sugar, or combination thereof; do.

A liquid formulation according to any one of the preceding embodiments, wherein said liquid formulation further comprises one or more ingredients selected from the group consisting of sugars or sugar alcohols, nonionic surfactants and amino acids. Characterized by

A liquid formulation according to any one of the preceding embodiments, wherein said liquid formulation is a liquid formulation of a long acting conjugate of a glucagon derivative peptide further comprising a sugar or sugar alcohol, an amino acid or both. and

A liquid formulation according to any one of the preceding embodiments, characterized in that it comprises a buffer substance, a sugar or sugar alcohol and an amino acid.

A liquid formulation according to any one of the preceding embodiments, characterized in that it comprises a buffer substance, a sugar or sugar alcohol, a non-ionic surfactant and an amino acid.

A liquid formulation according to any one of the preceding embodiments, characterized in that said liquid formulation further comprises a non-ionic surfactant, an amino acid, or both.

A liquid formulation according to any one of the preceding embodiments, characterized in that it comprises i) a sugar or sugar alcohol, ii) a non-ionic surfactant, or a combination thereof.

A liquid formulation according to any one of the preceding embodiments, characterized in that said glucagon derivative peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-11 and 13-45.

The peptide includes the amino acid sequences of SEQ ID NOs: 2 to 11, 13 to 45, or consists (essentially) of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11, 13 to 45. Good, but not limited to this.

Examples of such peptides may include, but particularly It is not limited. As another example, said peptide may be a peptide comprising, or consisting (essentially) of, an amino acid sequence selected from the group consisting of SEQ ID NO: 37, but is not particularly limited thereto. do not have.

A liquid formulation according to any one of the preceding embodiments, wherein said L is polyethylene glycol.

A liquid formulation according to any one of the foregoing embodiments, characterized in that the formula weight of the ethylene glycol repeating unit moiety within said L is in the range of 1-100 kDa.

A liquid formulation according to any one of the foregoing embodiments, wherein the structure of formula (1) is the structure of formula (2):

・・・（２）

... (2)

Here, X and F are as defined in chemical formula (1).

A liquid formulation according to any _one of _the preceding embodiments, wherein said ethylene glycol repeat unit is [ _OCH2CH2 ]n, n is a natural number, and [ _OCH2CH2 ]n sites within said peptide conjugate is determined to have an average molecular weight, for example, a number average molecular weight of 1 to 100 kDa.

A liquid formulation according to any one of the preceding embodiments, wherein the value of n is such that the average molecular weight of the [OCH ₂ CH ₂ ]n sites within the peptide conjugate is, for example, a number average molecular weight of 10 kDa. characterized by being defined

A liquid formulation according to any one of the preceding embodiments, characterized in that said X is C-terminally amidated.

A liquid formulation according to any one of the foregoing embodiments, characterized in that said X is linked through a cysteine sulfur atom within the peptide.

A liquid formulation according to any one of the preceding embodiments, characterized in that said immunoglobulin Fc segment is derived from IgG4.

A liquid formulation according to any one of the foregoing embodiments, wherein F is a structure in which two polypeptide chains are linked by disulfide bonds, and only through the nitrogen atoms of one of the two chains It is characterized by being connected.

A liquid formulation according to any one of the preceding embodiments, characterized in that said F comprises a monomer that is the amino acid sequence of SEQ ID NO:51.

A liquid formulation according to any one of the preceding embodiments, characterized in that said F is a homodimer of the monomers of the amino acid sequence of SEQ ID NO:51.

A liquid formulation according to any one of the foregoing embodiments, characterized in that said F is linked through the nitrogen atom of its N-terminal proline.

A liquid formulation according to any one of the preceding embodiments, characterized in that said immunoglobulin Fc segments F and X are non-glycosylated.

The liquid formulation according to the above embodiments, characterized in that the liquid formulation does not further comprise one or more ingredients selected from the group consisting of nonionic surfactants and amino acids.

The liquid formulation according to any one of the preceding embodiments, wherein said buffer substance is selected from the group consisting of citric acid and salts thereof, acetic acid and salts thereof, histidine and salts thereof, phosphoric acid and salts thereof and combinations thereof. It is characterized by being selected.

A liquid formulation according to any one of the preceding embodiments, wherein said buffer substance is acetic acid and its salts.

A liquid formulation according to any one of the preceding embodiments, characterized in that the pH of said liquid formulation is between 4.8 and 6.5.

A liquid formulation according to any one of the preceding embodiments, characterized in that the pH of said liquid formulation is between 4.8 and 6.0.

A liquid formulation according to any one of the preceding embodiments, characterized in that the pH of said liquid formulation is between 4.8 and 5.5.

A liquid formulation according to any one of the preceding embodiments, characterized in that the concentration of said buffering substance is between 5 and 100 mM for maintaining the pH of the liquid formulation between 4.8 and 6.5. do.

A liquid formulation according to any one of the foregoing embodiments, characterized in that said sugar or sugar alcohol is one or more selected from the group consisting of sucrose, mannitol and sorbitol.

A liquid formulation according to any one of the preceding embodiments, characterized in that said sugar or sugar alcohol is present in the liquid formulation at a concentration of 1-20% (w/v).

A liquid formulation according to any one of the preceding embodiments, wherein said long-acting conjugate is present in the formulation at 18-2807 nmol/mL, 18-936 nmol/mL, 90-562 nmol/mL, 187-562 nmol/mL, 187 It is characterized by being present at a concentration of ˜2807 nmol/mL, or 93-936 nmol/mL.

A liquid formulation according to any one of the preceding embodiments, wherein said sugar is glucose, fructose, galactose, lactose, maltose, sucrose, or a combination thereof.

A liquid formulation according to any one of the preceding embodiments, characterized in that said sugar is sucrose.

A liquid formulation according to any one of the preceding embodiments, characterized in that said sugar is present in a concentration of 3-15% (w/v).

A liquid formulation according to any one of the preceding embodiments, wherein said sugar alcohol is one or more selected from the group consisting of mannitol and sorbitol.

A liquid formulation according to any one of the preceding embodiments, characterized in that said liquid formulation further comprises one or more ingredients selected from the group consisting of non-ionic surfactants and amino acids.

A liquid formulation according to the preceding embodiment, characterized in that said liquid formulation does not contain a tonicity agent.

A liquid formulation according to any one of the preceding embodiments, wherein said nonionic surfactant is a poloxamer, polysorbate, or a combination thereof.

The liquid formulation according to any one of the preceding embodiments, wherein said nonionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. characterized by

A liquid formulation according to any one of the preceding embodiments, characterized in that said nonionic surfactant is present in the formulation at a concentration of 0.001-0.5% (w/v). do.

A liquid formulation according to any one of the preceding embodiments, characterized in that said amino acids are selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine and combinations thereof.

A liquid formulation according to any one of the preceding embodiments, characterized in that said amino acid is present in the formulation at a concentration of 0.01-1 mg/mL.

A liquid formulation according to any one of the foregoing embodiments, wherein said liquid formulation comprises 90-562 nmol/mL of the peptide conjugate of formula (1); said liquid formulation has a pH of 4.8-6.5. 5-25 mM buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts and combinations thereof; 1-20% (w/v) sugar alcohols; sugars, or combinations thereof; and 0.01-0.1% (w/v) of nonionic surfactants selected from poloxamers, polysorbates, or combinations thereof; and methionine, arginine, histidine, glycine, cysteine, characterized by containing 0.01-1 mg/mL of a stabilizer selected from the group consisting of lysine and combinations thereof.

A liquid formulation according to any one of the foregoing embodiments, wherein said liquid formulation comprises 90-562 nmol/mL of the peptide conjugate of formula (1); said liquid formulation has a pH of 4.8-6.5. 5-25 mM buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts and combinations thereof; 4-10% (w/v) sugar alcohols; sugars, or combinations thereof; and 0.01-0.1% (w/v) of nonionic surfactants selected from poloxamers, polysorbates, or combinations thereof; and methionine, arginine, histidine, glycine, cysteine, characterized by containing 0.01-1 mg/mL of a stabilizer selected from the group consisting of lysine and combinations thereof.

A liquid formulation according to any one of the foregoing embodiments, wherein said liquid formulation comprises 90-562 nmol/mL of the peptide conjugate of formula (1); said liquid formulation has a pH of 4.8-6.0. 5-25 mM buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts and combinations thereof; 4-10% (w/v) sugar alcohols; sugars, or combinations thereof; and 0.01-0.1% (w/v) of nonionic surfactants selected from poloxamers, polysorbates, or combinations thereof; and methionine, arginine, histidine, glycine, cysteine, characterized by containing 0.01-1 mg/mL of a stabilizer selected from the group consisting of lysine and combinations thereof.

A liquid formulation according to any one of the foregoing embodiments, wherein said liquid formulation comprises 90-562 nmol/mL of the peptide conjugate of formula (1); said liquid formulation has a pH of 4.8-5.5. 5-25 mM buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts and combinations thereof; 4-10% (w/v) sugar alcohols; sugars, or combinations thereof; and 0.01-0.1% (w/v) of nonionic surfactants selected from poloxamers, polysorbates, or combinations thereof; and methionine, arginine, histidine, glycine, cysteine, characterized by containing 0.01-1 mg/mL of a stabilizer selected from the group consisting of lysine and combinations thereof.

A liquid formulation according to any one of the preceding embodiments, said formulation having a pH in the range of 4.8 to 6.5; a buffer substance; a sugar selected from the group consisting of sucrose, mannitol, sorbitol and combinations thereof. or a sugar alcohol; an amino acid selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof; and polysorbate as a nonionic surfactant.

A liquid formulation according to any one of the foregoing embodiments, wherein in said glucagon derivative peptide long-acting conjugate, said glucagon derivative peptide comprises an amino acid sequence of general formula 1:

Y-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T- X30 (general formula 1, SEQ ID NO: 46)

In the general formula 1,
X2 is α-methyl-glutamic acid, Aib (aminoisobutylic acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S) or D-serine;
X7 is threonine (T), valine (V) or cysteine (C);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X13 is tyrosine (Y) or cysteine (C);
X14 is leucine (L) or cysteine (C);
X15 is aspartic acid (D), glutamic acid (E) or cysteine (C);
X16 is glutamic acid (E), aspartic acid (D), serine (S), α-methylglutamic acid, or cysteine (C), or absent;
X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C), or valine (V), or absent ;
X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), or cysteine (C), or absent;
X19 is alanine (A), arginine (R), serine (S), valine (V), cysteine (C), or absent;
X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), α-methyl-glutamic acid, or cysteine (C); figure;
X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine (C), or absent;
X23 is isoleucine (I), valine (V), arginine (R), or absent;
X24 is valine (V), arginine (R), alanine (A), cysteine (C), glutamic acid (E), lysine (K), glutamine (Q), α-methyl-glutamic acid, or leucine (L) or does not exist;
X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or absent;
X28 is glutamine (Q), lysine (K), asparagine (N), arginine (R), or absent;
X30 is cysteine (C) or absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

A liquid formulation according to any one of the preceding embodiments, wherein in said general formula 1, X2 is serine (S) or Aib (aminoisobutylic acid);
X7 is threonine (T), valine (V) or cysteine (C);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X13 is tyrosine (Y) or cysteine (C);
X14 is leucine (L) or cysteine (C);
X15 is aspartic acid (D) or cysteine (C);
X16 is glutamic acid (E), serine (S) or cysteine (C);
X17 is aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C), or valine (V);
X18 is aspartic acid (D), glutamic acid (E), arginine (R), or cysteine (C);
X19 is alanine (A) or cysteine (C),
X20 is glutamine (Q), aspartic acid (D), lysine (K), or cysteine (C);
X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine (C);
X23 is isoleucine (I), valine (V) or arginine (R);
X24 is valine (V), arginine (R), alanine (A), glutamic acid (E), lysine (K), glutamine (Q), or leucine (L);
X27 is isoleucine (I), valine (V), alanine (A), methionine (M), glutamine (Q) or arginine (R);
X28 is glutamine (Q), lysine (K), asparagine (N) or arginine (R);
X30 is cysteine (C) or absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

A liquid formulation according to any one of the preceding embodiments,
In the general formula 1, X2 is serine (S) or Aib (aminoisobutylic acid);
X7 is cysteine (C), threonine (T), or valine (V);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X13 is tyrosine (Y) or cysteine (C);
X14 is leucine (L) or cysteine (C);
X15 is aspartic acid (D) or cysteine (C);
X16 is glutamic acid (E), serine (S) or cysteine (C);
X17 is glutamic acid (E), lysine (K), arginine (R), cysteine (C), or valine (V);
X18 is arginine (R) or cysteine (C);
X19 is alanine (A) or cysteine (C),
X20 is glutamine (Q) or lysine (K);
X21 is aspartic acid (D), glutamic acid (E), valine (V), or cysteine (C);
X23 is valine (V),
X24 is valine (V) or glutamine (Q);
X27 is methionine (M);
X28 is asparagine (N) or arginine (R);
X30 is cysteine (C) or absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

A liquid formulation according to any one of the preceding embodiments,
In the general formula 1, X2 is Aib (aminoisobutylic acid);
X7 is cysteine (C), threonine (T), or valine (V);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K);
X13 is tyrosine (Y) or cysteine (C);
X14 is leucine (L) or cysteine (C);
X15 is aspartic acid (D) or cysteine (C);
X16 is glutamic acid (E), serine (S) or cysteine (C);
X17 is lysine (K), arginine (R), cysteine (C), or valine (V);
X18 is arginine (R) or cysteine (C);
X19 is alanine (A) or cysteine (C),
X20 is glutamine (Q) or lysine (K);
X21 is aspartic acid (D), glutamic acid (E), or cysteine (C);
X23 is valine (V),
X24 is glutamine (Q);
X27 is methionine (M);
X28 is asparagine (N) or arginine (R);
X30 is cysteine (C) or absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

A liquid formulation according to any one of the preceding embodiments,
In the general formula 1, X2 is serine (S) or Aib (aminoisobutylic acid);
X7 is threonine (T), valine (V) or cysteine (C);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X13 is tyrosine (Y) or cysteine (C);
X14 is leucine (L) or cysteine (C);
X15 is aspartic acid (D) or cysteine (C);
X16 is glutamic acid (E), serine (S) or cysteine (C);
X17 is aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C), or valine (V);
X18 is aspartic acid (D), glutamic acid (E), arginine (R), or cysteine (C);
X19 is alanine (A) or cysteine (C),
X20 is glutamine (Q), aspartic acid (D), or lysine (K);
X21 is aspartic acid (D) or glutamic acid (E);
X23 is valine (V),
X24 is valine (V) or glutamine (Q);
X27 is isoleucine (I) or methionine (M);
X28 is asparagine (N) or arginine (R);
X30 is cysteine (C) or absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

A liquid formulation according to any one of the preceding embodiments,
In the general formula 1, X2 is Aib (aminoisobutylic acid);
X7 is threonine (T);
X10 is tyrosine (Y);
X12 is lysine (K);
X13 is tyrosine (Y);
X14 is leucine (L),
X15 is aspartic acid (D) or cysteine (C);
X16 is glutamic acid (E), serine (S) or cysteine (C);
X17 is lysine (K) or arginine (R);
X18 is arginine (R);
X19 is alanine (A);
X20 is glutamine (Q), cysteine (C), or lysine (K);
X21 is aspartic acid (D), cysteine (C), valine (V) or glutamic acid (E);
X23 is valine (V) or arginine (R);
X24 is glutamine (Q) or leucine (L);
X27 is methionine (M);
X28 is asparagine (N) or arginine (R);
Characterized by the absence of X30. (However, cases where the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12 are excluded).

A liquid formulation according to any one of the foregoing embodiments, wherein in said glucagon derivative peptide long-acting conjugate, said glucagon derivative peptide comprises an amino acid sequence of general formula 2:

Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-NT- X30 (general formula 2, SEQ ID NO: 47)

In the general formula 2,
X7 is threonine (T), valine (V) or cysteine (C);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X15 is aspartic acid (D) or cysteine (C);
X16 is glutamic acid (E) or serine (S);
X17 is lysine (K) or arginine (R);
X20 is glutamine (Q) or lysine (K);
X21 is aspartic acid (D) or glutamic acid (E);
X24 is valine (V) or glutamine (Q);
X30 is cysteine (C) or absent (except when the amino acid sequence of general formula 2 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

A liquid formulation according to any one of the preceding embodiments, characterized in that said glucagon derivative peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-45.

A liquid formulation according to any one of the preceding embodiments, characterized in that said glucagon derivative peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15 and 36-44.

A liquid formulation according to any one of the preceding embodiments, characterized in that said glucagon derivative peptide comprises the amino acid sequence of SEQ ID NO:37.

A liquid formulation according to any one of the preceding embodiments, wherein said glucagon derivative peptide has a pI different from the pI of native glucagon of 6.8, such as a pI of 6.5 or less or a pI of 7.0 or more. It is characterized by

A liquid formulation according to any one of the preceding embodiments, characterized in that said glucagon derivative peptide has a carboxy group (COOH) at its C-terminus.

A liquid formulation according to any one of the preceding embodiments, characterized in that said glucagon derivative peptide is a derivative of native glucagon that activates the glucagon receptor.

A liquid formulation according to any one of the foregoing embodiments, wherein in said long-acting conjugate of said glucagon derivative peptide, said glucagon derivative peptide has a pI of 6-7 and a relative in vitro activity of 200 relative to native glucagon. % or more.

A liquid formulation according to any one of the foregoing embodiments, wherein the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24 and X24 and X28 of general formula 1 or 2 are It is characterized in that each amino acid of at least one amino acid pair is replaced with glutamic acid or lysine capable of forming a ring.

A liquid formulation according to any one of the foregoing embodiments, wherein each of the amino acids of the amino acid pair X12 and X16, the amino acid pair X16 and X20, or the amino acid pair X17 and X21 of general formula 1 or 2 is a cyclic is substituted with glutamic acid or lysine that can form

A liquid formulation according to any one of the foregoing embodiments, wherein in said general formula 1 or 2, the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24 and X24 and X28 A ring is formed between each amino acid in at least one amino acid pair of .

A liquid formulation according to any one of the preceding embodiments, characterized in that said ring is a lactam ring.

A liquid formulation according to any one of the above embodiments, wherein in the general formula 1 or 2, X16 is glutamic acid, X20 is lysine, and the side chains of X16 and X20 form a lactam ring. It is characterized by

A liquid formulation according to any one of the preceding embodiments, characterized in that said immunoglobulin Fc segment is an Fc segment derived from IgG, IgA, IgD, IgE or IgM.

A liquid formulation according to any one of the preceding embodiments, wherein said immunoglobulin Fc segment comprises (a) CH1, CH2, CH3 and CH4 domains; (b) CH1 and CH2 domains; (c) CH1 (d) CH2 and CH3 domains; (e) one or more domains in CH1, CH2, CH3 and CH4 domains with an immunoglobulin hinge region or part of a hinge region; combination; and (f) selected from the group consisting of a dimer of each domain of a heavy chain constant region and a light chain constant region.

A liquid formulation according to any one of the preceding embodiments, wherein each domain of said immunoglobulin Fc segment is an immunoglobulin-derived variant selected from the group consisting of IgG, IgA, IgD, IgE and IgM. It is characterized as being a hybrid of domains that have origins in

A liquid formulation according to any one of the preceding embodiments, wherein said immunoglobulin Fc segment is in dimeric or multimeric form, composed of single-chain immunoglobulins consisting of domains of the same origin. Characterized by

A liquid formulation according to any one of the preceding embodiments, characterized in that said immunoglobulin Fc segment is an IgG4 Fc segment.

A liquid formulation according to any one of the preceding embodiments, characterized in that said immunoglobulin Fc segment is a human non-glycosylated IgG4 Fc segment.

A liquid preparation according to any one of the above-described specific examples, wherein the immunoglobulin Fc fragment is a variant in which a site capable of forming a disulfide bond is removed, or a variant in which a part of amino acids at the N-terminus is removed from the native Fc. , a variant in which a methionine residue is added to the N-terminus of the native Fc, a variant in which the complement binding site is removed, or a variant in which the ADCC (antibody dependent cell mediated cytotoxicity) site is removed, or the variant It is characterized as being a derivative of native Fc, including combinations.

Another aspect embodying the invention is a method of making the liquid formulation.

As one embodiment, the present invention provides (i) a long-acting conjugate of a glucagon derivative peptide, wherein the glucagon derivative peptide and an immunoglobulin Fc segment are linked together, and (ii) (a) a buffer substance and (b) a sugar alcohol, A method of making a liquid formulation comprising mixing sugar or a combination thereof.

The method according to the preceding embodiment, wherein said stabilizing agent further comprises one or more ingredients selected from the group consisting of sugars or sugar alcohols, nonionic surfactants, and amino acids.

A method according to any one of the preceding embodiments, wherein said liquid formulation is a liquid formulation of a long-acting conjugate of a glucagon derivative peptide further comprising a sugar or sugar alcohol, an amino acid, or both. do.

A method according to any one of the preceding embodiments, characterized in that said liquid formulation comprises a buffer substance, a sugar or sugar alcohol and an amino acid.

A method according to any one of the preceding embodiments, characterized in that said liquid formulation comprises a buffer substance, a sugar or sugar alcohol, a nonionic surfactant and an amino acid.

Detailed contents for carrying out the present invention are as follows.

It should be noted that each description and embodiment disclosed in this application also applies to other descriptions and embodiments, respectively. That is, the present invention includes all combinations of various elements disclosed in this application. Moreover, the present invention is not limited to the following specific description.

In addition, those skilled in the art will be able to recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the invention described in this application. Also, such equivalents are intended to be included in this invention.

Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as Aib (α-aminoisobutyric acid, 2-aminoisobutylic acid), Sar (N-methylglycine), α- The generally accepted three-letter code for other amino acids such as methylglutamic acid (α-methyl-glutamic acid) is used. Also, amino acids referred to as abbreviations herein are described by the IUPAC-IUB nomenclature.

Alanine Ala, A Arginine Arg, R
Asparagine Asn, N Aspartic acid Asp, D
Cysteine Cys, C Glutamic acid Glu, E
Glutamine Gln, Q Glycine Gly, G
Histidine His, H Isoleucine Ile, I
Leucine Leu, L Lysine Lys, K
Methionine Met, M Phenylalanine Phe, F
Proline Pro, P Serine Ser, S
Threonine Thr, T Tryptophan Trp, W
Tyrosine Tyr, Y Valine Val, V

One aspect embodying the present invention provides liquid formulations of long-acting conjugates of glucagon derivatives.

Specifically, the present invention comprises a pharmacologically effective amount of a long-acting conjugate of a glucagon derivative peptide, in which a glucagon derivative peptide and an immunoglobulin Fc segment are linked to each other, and a buffer substance and a sugar alcohol, sugar, or Liquid formulations of long-acting conjugates of glucagon derivative peptides, including combinations.

Specifically, the present invention provides a liquid formulation comprising a long-acting conjugate of formula (1) below; a buffering substance; and a sugar alcohol, sugar, or a combination thereof:

X-La-F (1)

In the chemical formula (1), X is a glucagon derivative peptide,
L is a linker,
a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
F is the immunoglobulin Fc segment;
- indicates a covalent bond:

[General formula 1]
Y-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T- X30 (general formula 1, SEQ ID NO: 46)

In the general formula 1,
X2 is α-methyl-glutamic acid, Aib (aminoisobutylic acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S) or D-serine;
X7 is threonine (T), valine (V) or cysteine (C);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X13 is tyrosine (Y) or cysteine (C);
X14 is leucine (L) or cysteine (C);
X15 is aspartic acid (D), glutamic acid (E) or cysteine (C);
X16 is glutamic acid (E), aspartic acid (D), serine (S), α-methylglutamic acid, or cysteine (C), or absent;
X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C), or valine (V), or absent ;
X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), or cysteine (C), or absent;
X19 is alanine (A), arginine (R), serine (S), valine (V), cysteine (C), or absent;
X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), α-methylglutamic acid, or cysteine (C), or absent ;
X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine (C), or absent;
X23 is isoleucine (I), valine (V), arginine (R), or absent;
X24 is valine (V), arginine (R), alanine (A), cysteine (C), glutamic acid (E), lysine (K), glutamine (Q), α-methyl-glutamic acid, or leucine (L) or does not exist;
X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or absent;
X28 is glutamine (Q), lysine (K), asparagine (N), arginine (R), or absent;
X30 is cysteine (C) or absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

In the present invention, the term "liquid formulation" means a liquid formulation of a pharmaceutical form, and includes both liquid formulations for internal use and for external use.

The liquid preparation of the present invention comprises a long-acting conjugate of formula (1) having a pharmacological effect and a substance capable of stably maintaining and/or preserving the pharmacologically effective substance for a certain period of time when it is formulated in a liquid form. include. Ingredients other than the long-acting conjugate of formula (1) exhibiting a pharmacological effect in the liquid formulation may be mixed with a stabilizer.

In the liquid formulation of the long-acting conjugate of formula (1) of the present invention, storage stability is important to ensure accurate dosage.

The present invention provides a specific concentration of the long-acting conjugate of chemical formula (1), which is a substance exhibiting a pharmacological effect; 20% (w/v) of sugar alcohol, sugar or a combination thereof; began to provide form.
The concentration of the long-acting glucagon derivative peptide contained in the liquid formulation of the present invention is about 18 to about 2810 nmol/mL, about 18 to about 2807 nmol/mL, about 18 to about 2623 nmol/mL, about 18 to about 2436 nmol. /mL, about 18 to about 2248 nmol/mL, about 18 to about 2061 nmol/mL, about 18 to about 1874 nmol/mL, about 18 to about 1686 nmol/mL, about 18 to about 1499 nmol/mL, about 18 to about 1312 nmol/mL About 18 to about 570 nmol/mL, about 18 to about 562 nmol/mL, about 18 to about 469 nmol/mL, about 18 to about 375 nmol/mL, about 18 to about 281 nmol/mL, about 18 to about 188 nmol/mL, about 18 to about about 94 nmol/mL, about 187 nmol/mL, about 188 nmol/mL, about 187.09 nmol/mL, about 187.1 nmol/mL, about 93 to about 188 nmol/mL, about 93 to about 281 nmol/mL, about 93 to about 375 nmol /mL, about 93 to about 469 nmol/mL, about 93 to about 562 nmol/mL, about 93 to about 656 nmol/mL, about 93 to about 750 nmol/mL, about 93 to about 843 nmol/mL, about 93 to about 940 nmol/mL , about 93 to about 936 nmol/mL, about 187 to about 281 nmol/mL, about 187 to about 375 nmol/mL, about 187 to about 469 nmol/mL, about 187 to about 570 nmol/mL, or about 187 to about 562 nmol/mL. may be used, but is not limited to this. As one specific example, the concentration of the long-acting conjugate may range from 18 to 936 nmol/mL, but is not limited thereto.

In the present invention, the term "stabilizer" refers to a substance that stabilizes a constituent component such as an active ingredient in a formulation for a certain period of time.

Preferably, the stabilizer of the present invention does not contain albumin. Human serum albumin, which is used as a protein stabilizer, is manufactured from human blood and has the potential for contamination with human-derived pathogenic viruses. may induce an allergic reaction to Since the albumin-free stabilizer of the present invention does not contain foreign proteins such as human- or animal-derived serum albumin or purified gelatin, there is little risk of virus infection.

In the present invention, the stabilizing agent particularly means a substance that enables the long-acting conjugate of the glucagon derivative to be stably stored. In long-acting conjugates of glucagon derivatives, storage stability is important not only for ensuring accurate dosage, but also for suppressing potential generation of antigenic substances to the glucagon derivative.

The buffer substance, which is one component contained in the liquid preparation of the present invention, maintains the pH of the solution so that the pH of the liquid preparation does not change abruptly so that the long-acting conjugate of formula (1) is stable. can be done. The buffer substance is also called a buffer system, and the buffer substance or buffer system serves to maintain the pH of the liquid formulation. Any buffering substance that maintains a pH at which the long-acting conjugate of formula (1), which is the target substance to be stabilized, can be stabilized may be used without limitation.

The buffer substances include phosphoric acid and its conjugate base alkali salts (e.g., phosphates: sodium phosphate, potassium phosphate, or hydrogen or dihydrogen salts thereof), citric acid and its salts (e.g., sodium citrate). ), acetic acid and its salts (eg, sodium acetate), histidine and its salts, and mixtures of these buffering substances, including but not limited to.

The liquid formulation of the present invention contains a buffer solution containing the buffer substance as a solvent for the liquid formulation. , sodium acetate buffer), phosphate buffer (e.g., sodium phosphate buffer), histidine buffer, and combinations thereof; buffer substances (citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, or combinations thereof) at concentrations sufficient to maintain the pH of the target liquid formulation may be included.

The pH of the liquid formulation is from about 4.6 to about pH 6.5, such as from about pH 4.8 to about pH 6.5, from about pH 4.8 to about 6.4, from about pH 4.8 to about 6.3, pH about 4.8 to about pH 6.2, pH about 4.8 to about 6.1, pH about 4.8 to about 6.0, pH about 4.8 to about 5.9, pH about 4.8 to about 5.8, pH about 4.8 to 5.7, pH about 4.8 to about 5.6, pH about 4.8 to 5.5, pH about 4.8 to about 5.5. 4, pH about 4.8 to about 5.3, pH about 4.8 to about 5.2, pH about 4.8 to about 5.1, pH about 4.9 to about pH 6.5, pH about 4 .9 to about 6.4, pH about 4.9 to about 6.3, pH about 4.9 to about pH 6.2, pH about 4.9 to about 6.1, pH about 4.9 to about 6 pH about 4.9 to about 5.9, pH about 4.9 to about 5.8, pH about 4.9 to 5.7, pH about 4.9 to about 5.6, pH about 4.0. 9 to 5.5, pH about 4.9 to about 5.4, pH about 4.9 to about 5.3, pH about 4.9 to about 5.2, pH about 4.9 to about 5.1, or pH about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, or 6.5, but is not particularly limited thereto.

The concentration of the liquid formulation that allows the target pH to be reached may be from about 1 mM to about 200 mM, more specifically from about 5 mM to about 100 mM, from about 5 mM to about 80 mM, from about 5 mM to about 40 mM, about 8 mM to about 40 mM, about 5 mM to about 30 mM, or about 5 mM to about 25 mM, about 10 mM to about 25 mM, about 15 mM to about 25 mM, about 18 mM to about 24 mM, about 18 mM to about 22 mM, or about 20 mM. However, it is not particularly limited to this.

As one example, the buffer substance may be acetic acid and its salts, but is not limited thereto.

In another embodiment, the buffer solution may be, but is not limited to, an acetate buffer solution (e.g. sodium acetate buffer solution) or a citrate buffer solution (e.g. sodium citrate buffer solution). isn't it.

On the other hand, in the manufacture of formulations, the ingredients can be dissolved in water (e.g., WFI) and the pH of the buffer solution or formulation adjusted to the desired pH using HCl and/or NaOH, etc., which is well known in the art. This is the method already commonly used. Therefore, even if there is no separate mention of a pH adjuster in the claims, it can be understood by those skilled in the art that the formulation can have an adjusted pH through such a method.

The sugar alcohol, which is one of the constituents contained in the stabilizer of the present invention, refers to a substance containing a large number of hydroxyl groups, including substances substituted with alcohol groups such as sugar aldehyde groups and/or ketone groups, and multiple hydroxyl groups. It also contains sugars and the like. Said sugars or sugar alcohols can increase the stability of long-acting conjugates of glucagon derivatives. For example, the sugar alcohol may be one or more selected from the group consisting of mannitol and sorbitol, but is not limited thereto.

Saccharide, which is one component contained in the liquid preparation of the present invention, refers to monosaccharides, disaccharides, polysaccharides, oligosaccharides, etc. can increase the stability of long-acting conjugates of peptides that have activity in Specific examples include monosaccharides such as mannose, glucose, fructose, galactose, fucose and xylose; disaccharides such as lactose, maltose and sucrose; polysaccharides such as raffinose and dextran. Not restricted.

In one embodiment, the sugar can be glucose, fructose, galactose, lactose, maltose, sucrose, or combinations thereof, but is not limited thereto.

For example, the sugar may be sucrose, but is not particularly limited to this.

The sugar alcohol, sugar, or combination thereof is about 0.5 to about 20% (w/v), about 0.5 to about 15% (w/v), about 0.5 to about 10% (w/v), about 0.5 to about 8% (w/v), about 1 to about 20% (w/v), about 1-15% (w/v), about 1- 10% (w/v), about 1-8% (w/v), about 2-about 15% (w/v), about 2-about 12% (w/v), about 2-about 12% ( w/v), about 3 to about 10% (w/v), about 4 to about 10% (w/v), about 4 to about 8% (w/v), about 4 to about 6% (w/v) v), about 5 to about 10% (w/v), about 6 to about 10% (w/v), about 7 to about 10% (w/v), about 7 to about 9% (w/v) , about 8 to about 9% (w/v), or about 1.0% (w/v), about 3.0% (w/v), about 5.0% (w/v), or about 8 It may be present at a concentration of, but not limited to, .0% (w/v). In addition, although not particularly limited thereto, the liquid formulation may further contain one or more components selected from the group consisting of nonionic surfactants and amino acids.

Thus, the stabilizing agent of said liquid formulation may consist essentially of i) a buffer substance and ii) a sugar or sugar alcohol, but i) a buffer substance, ii) a sugar or sugar alcohol, and iii) a non-ionic surfactants; i) buffer substances, ii) sugars or sugar alcohols, iii) amino acids; and iv) nonionic surfactants; i) buffer substances, ii) sugars or sugar alcohols, and iii) amino acids; Substances ii) sugars or sugar alcohols, iii) nonionic surfactants, and iv) amino acids may essentially consist of, but are not limited to. Here, it is clear that the type and concentration or pH of each component constituting the stabilizer are all applied to the contents described above and below.

Although not particularly limited thereto, the nonionic surfactant, which is one component contained in the liquid preparation, can reduce the surface tension of the protein solution to prevent the adsorption or aggregation of the protein on the hydrophobic surface. can.

Specific examples of nonionic surfactants used in the present invention include polysorbates (e.g., polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monolaurate), palmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate); -(CH2CH2O)-)), poloxamer (PEO-PPO-PEO copolymer; PEO: poly (ethylene oxide), PPO: poly (propylene oxide)), polyethylene-polypropylene glycol, polyoxyethylene compound (For example, polyoxyethylene-stearate, polyoxyethylene alkyl ether (alkyl: C1-C30), polyoxyethylene monoallyl ether, alkylphenyl polyoxyethylene copolymer (alkyl: C1-C30), etc.), sodium dodecyl sulfate (sodium dodecyl sulfate, SDS), etc., or polysorbate or poloxamer can be mentioned, and these can be used in the form of one or a combination of two or more.

Specifically, the nonionic surfactant may be polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, or poloxamer 188, which may be used in combination, but particularly It is not limited.

In the present invention, the nonionic surfactant is preferably not contained at a high concentration, specifically, the concentration of about 0.2% (w/v) or less, for example, about 0.2% (w/v) or less, in the formulation of the present invention. 001 to about 0.2% (w/v), about 0.001 to about 0.1% (w/v), about 0.001 to about 0.05% (w/v), about 0.005 to about 0.08% (w/v), about 0.002 to about 0.05% (w/v), about 0.005 to about 0.05% (w/v), about 0.01 to about 0 .05% (w/v), about 0.01 to about 0.04% (w/v), about 0.01 to about 0.03% (w/v), about 0.01 to about 0.1 % (w/v), or about 0.02% (w/v), but is not specifically limited thereto.

Amino acids that are one type of stabilizing agent as an optional component that can be added to the liquid formulation may be, but are not limited to, methionine, arginine, histidine, glycine, cysteine, lysine, or combinations thereof.

The amino acid can suppress the generation of impurities that may occur due to protein oxidation reaction, etc., but is not particularly limited thereto.

The amino acid is present in the formulation at a concentration of about 0.01 to about 1 mg/mL, about 0.01 to about 0.8 mg/mL, about 0.01 to about 0.5 mg/mL, about 0.02 to about 0.5 mg/mL. It may be present at, but is not limited to, 5 mg/mL, or from about 0.02 to about 0.4 mg/mL, or about 0.1 mg/mL.

As one specific example, the liquid preparation containing a buffer substance and a sugar or sugar alcohol does not contain a tonicity agent, or contains one or more components selected from the group consisting of a nonionic surfactant and an amino acid. may not additionally include, but is not limited to,

On the other hand, the liquid preparation of the present invention contains sugar alcohol, sugar, or a combination thereof, which are essential constituents of the liquid preparation; In addition, other components or materials known in the art may be optionally included as long as they do not impair the effects of the present invention, but are not limited thereto.

Meanwhile, the formulation may further contain a polyhydric alcohol, but is not particularly limited thereto.

For example, i) a buffer substance and ii) a sugar or sugar alcohol as well as a polyhydric alcohol, i) a buffer substance, ii) a sugar or sugar alcohol, and iii) a nonionic surfactant; i) a buffer substance, ii) a sugar or sugar alcohol, iii) an amino acid; and iv) a nonionic surfactant; i) a buffer substance, ii) a sugar or sugar alcohol, and iii) an amino acid; Alternatively, the stabilizer consisting essentially of sugar alcohol, iii) nonionic surfactant, and iv) amino acid may further contain a polyhydric alcohol, but is not particularly limited thereto.

Examples of polyhydric alcohols that may be further included in the formulations of the present invention include propylene glycol and low molecular weight polyethylene glycols, glycerol, low molecular weight polypropylene glycols and the like, one or more of which are It is used in the form of a combination of, but is not limited to.

In the present invention, the long-acting conjugate of formula (1) is an active ingredient contained in the liquid formulation of the present invention, and may be contained in the formulation in a pharmacologically effective amount. Illustratively, the concentration of the long-acting conjugate is in the formulation from about 18 to about 2810 nmol/mL, from about 18 to about 2807 nmol/mL, from about 18 to about 2623 nmol/mL, from about 18 to about 2436 nmol/mL, from about 18 to about 2436 nmol/mL, from about 18 to about 2807 nmol/mL, about 2248 nmol/mL, about 18 to about 2061 nmol/mL, about 18 to about 1874 nmol/mL, about 18 to about 1686 nmol/mL, about 18 to about 1499 nmol/mL, about 18 to about 1312 nmol/mL, about 18 to about 1124 nmol /mL, about 18 to about 940 nmol/mL, about 18 to about 936 nmol/mL, about 18 to about 843 nmol/mL, about 18 to about 750 nmol/mL, about 18 to about 656 nmol/mL, about 18 to about 570 nmol/mL About 187 nmol/mL, about 188 nmol/mL, about 187.09 nmol/mL, about 187.1 nmol/mL, about 93 to about 188 nmol/mL, about 93 to about 281 nmol/mL, about 93 to about 375 nmol/mL, about 93 to about 469 nmol/mL, about 93 to about 562 nmol/mL, about 93 to about 656 nmol/mL, about 93 to about 750 nmol/mL, about 93 to about 843 nmol/mL, about 93 to about 940 nmol/mL, about 93 to about 936 nmol /mL, from about 187 to about 281 nmol/mL, from about 187 to about 375 nmol/mL, from about 187 to about 469 nmol/mL, from about 187 to about 570 nmol/mL or from about 187 to about 562 nmol/mL, but particularly It is not limited to this.

In the present invention, the term "about" is a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, ±0.01, etc., and the term about including, but not limited to, all numerical values in ranges equivalent or similar to the numerical values that follow.

As one specific example, the liquid preparation is a peptide conjugate of chemical formula (1); citric acid and its salts, acetic acid and its salts, histidine and sugars; and nonionic surfactants selected from poloxamers, polysorbates or combinations thereof; and methionine, arginine, histidine, glycine, cysteine, lysine. and combinations thereof.

As one specific example, the liquid preparation contains 90 to 562 nmol/mL of the peptide conjugate of chemical formula (1); and salts thereof, histidine and salts thereof, phosphoric acid and salts thereof, and combinations thereof; 1-20% (w/v) of sugar alcohols, sugars, or combinations thereof; and poloxamers. 0.01-0.1% (w/v) of a nonionic surfactant selected from , polysorbate or combinations thereof; and from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine and combinations thereof. It may also be a liquid formulation containing 0.01-1 mg/mL of the stabilizer of choice.

As one specific example, the liquid preparation contains 90 to 562 nmol/mL of the peptide conjugate of chemical formula (1); 5-25 mM buffer substances selected from and salts thereof, histidine and salts thereof, phosphate and salts thereof and combinations thereof; 4-10% (w/v) sugars; and poloxamers, polysorbates or combinations thereof. 0.01-0.1% (w/v) of a nonionic surfactant; It may be a liquid formulation containing mL of stabilizer.

On the other hand, the long-acting conjugate of glucagon derivative peptide, which is an active ingredient contained in the liquid preparation of the present invention, will be described below in more detail.

In the present invention, the term “long-acting conjugate of glucagon derivative” or “long-acting conjugate of glucagon derivative peptide” refers to a form in which an immunoglobulin Fc segment is linked to a glucagon derivative. Specifically, the conjugate may be a glucagon derivative covalently linked to an immunoglobulin Fc segment through a linker, but is not particularly limited thereto.

The conjugate can exhibit increased potency compared to the peptide to which the immunoglobulin Fc segment is not bound. In the present invention, the conjugate of the peptide of general formula 1 according to formula (1) is " long-acting conjugate" and may be mixed with "peptide conjugate" or "long-acting conjugate of formula (1)".

In one embodiment, the immunoglobulin Fc segment and X may be non-glycosylated, but are not limited to this.

Alternatively, such conjugates may be non-naturally occurring.

The long-acting conjugate of the present application may be in a form in which the glucagon derivative peptide and the immunoglobulin Fc segment are linked to each other, and the linking method is not particularly limited, but the peptide and the immunoglobulin Fc segment are linked to each other through a linker. It can be anything.

As one specific example, the long-acting conjugate of the present invention has the structure of the following chemical formula (1).

X-La-F

In the chemical formula (1), X is a peptide of the following general formula 1,
L is a linker containing ethylene glycol repeating units;
a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
F is the human immunoglobulin Fc segment;
- indicates a covalent bond.

X in the long-acting conjugate of formula (1) may be a peptide having activity against glucagon derivatives. A "peptide having activity against a glucagon derivative" includes a variety of substances, such as a variety of peptides, that have a significant level of activity against a glucagon derivative.

More specifically, the peptide having activity against glucagon derivatives is a peptide having activity against glucagon derivatives comprising the sequence of general formula 1 below:

[General formula 1]
Y-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T- X30 (general formula 1, SEQ ID NO: 46)

In the general formula 1,
X2 is α-methyl-glutamic acid, Aib (aminoisobutylic acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S) or D-serine;
X7 is threonine (T), valine (V) or cysteine (C);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X13 is tyrosine (Y) or cysteine (C);
X14 is leucine (L) or cysteine (C);
X15 is aspartic acid (D), glutamic acid (E) or cysteine (C);
X16 is glutamic acid (E), aspartic acid (D), serine (S), α-methylglutamic acid, or cysteine (C), or absent;
X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C), or valine (V), or absent ;
X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), or cysteine (C), or absent;
X19 is alanine (A), arginine (R), serine (S), valine (V), cysteine (C), or absent;
X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), α-methylglutamic acid, or cysteine (C), or absent ;
X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine (C), or absent;
X23 is isoleucine (I), valine (V), arginine (R), or absent;
whether X24 is valine (V), arginine (R), alanine (A), cysteine (C), glutamic acid (E), lysine (K), glutamine (Q), α-methylglutamic acid, or leucine (L) , does not exist;
X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or absent;
X28 is glutamine (Q), lysine (K), asparagine (N), arginine (R), or absent;
X30 may be cysteine (C) or may be absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

A liquid formulation according to any one of the foregoing embodiments, wherein in said glucagon derivative peptide long-acting conjugate, said glucagon derivative peptide comprises an amino acid sequence of general formula 2:

Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-NT- X30 (general formula 2, SEQ ID NO: 47)

In the general formula 2,
X7 is threonine (T), valine (V) or cysteine (C);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X15 is aspartic acid (D) or cysteine (C);
X16 is glutamic acid (E) or serine (S);
X17 is lysine (K) or arginine (R);
X20 is glutamine (Q) or lysine (K);
X21 is aspartic acid (D) or glutamic acid (E);
X24 is valine (V) or glutamine (Q);
X30 is cysteine (C) or absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

The peptide contains the amino acid sequences of SEQ ID NOs: 2 to 11 and 13 to 45, and even if it is (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11 and 13 to 45 Good, but not limited to this.

Examples of such peptides may include, but particularly It is not limited. As another example, said peptide may be a peptide comprising, or consisting (essentially) of, an amino acid sequence selected from the group consisting of SEQ ID NO: 37, but is not particularly limited thereto. do not have.

Also, the glucagon derivative is characterized by containing an intramolecular bridge. For example, it may be a covalent bridge or a non-covalent bridge, and in particular may be in a form comprising a ring. It may form a ring between the 16th amino acid glutamic acid and the 20th amino acid lysine, which are the underlined amino acid residues of general formula 1, but is not particularly limited thereto. Non-limiting examples of such rings may include lactam bridges (or lactam rings).

In addition, the peptide according to the present invention includes the peptide itself, a salt thereof (eg, a pharmaceutically acceptable salt of the peptide), or a solvate thereof. Also, the peptide may be in any pharmaceutically acceptable form.

The type of salt is not particularly limited. However, it is preferably in a form that is safe and effective for individuals such as mammals, but is not particularly limited to this.

The term "pharmaceutically acceptable" means a substance that can be effectively used for a desired purpose without causing undue toxicity, irritation, allergic reaction, etc., within the scope of pharmaceutical judgment. means.

As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric, bromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic , citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid and the like. Salts derived from suitable bases may include alkali metals such as sodium, potassium, alkaline earth metals such as magnesium, ammonium, and the like.

Also, the term "solvate" used in the present invention refers to a complex of the peptide according to the present invention or a salt thereof with a solvent molecule.

In addition, even if it is described as "a peptide composed of a specific SEQ ID NO" in the present application, if it has the same or corresponding activity as a peptide composed of the amino acid sequence of the SEQ ID NO, the amino acid sequence of the SEQ ID NO does not exclude meaningless sequence additions or naturally occurring mutations before or after the It is self-evident that it belongs to

Alternatively, such peptides may be non-naturally occurring.

The C-terminus of the peptide may be amidated, or may be a peptide having a free carboxyl group (--COOH), or may include a peptide in which the C-terminus is not modified, but is limited to this. not.

As one specific example, the X may have an amidated C-terminal, but is not limited to this.

As one specific example, the X may be non-glycosylated, but is not limited to this.

The peptide of general formula 1 can be synthesized through a solid phase synthesis method, can be produced by a recombinant method, and can be commercially commissioned, but is not limited thereto.
In the present invention, the term "long-acting conjugate of formula (1)" is an active ingredient contained in the liquid preparation of the present invention, and may be contained in the liquid preparation in a pharmacologically effective amount. Specifically, the peptide having activity against the glucagon derivative and the immunoglobulin Fc region are linked to each other by a linker, and the conjugate is active against the glucagon derivative to which the immunoglobulin Fc region is not bound. can exhibit increased persistence of efficacy compared to peptides with

In the long-acting conjugate of the chemical formula (1), the linkage between X, which is a peptide having activity against glucagon derivatives, and the immunoglobulin Fc segment may be a physical or chemical bond, or may be a non-covalent or covalent bond. It may be, specifically, a covalent bond, but is not limited to this.

In addition, the peptide conjugate of chemical formula (1) is not particularly limited in the method of linking X, which is a peptide having activity against glucagon derivatives, to the immunoglobulin Fc segment, but the glucagon receptor, GLP-1 receptor and GIP are linked through a linker. A peptide having activity against a receptor and an immunoglobulin Fc segment may be linked together.

Specifically, the long-acting conjugate contained in the liquid preparation of the present application may be represented by the chemical formula (1).

In Formula (1), X and F may be bonded to each other through L by a covalent bond.

More specifically, X and L, and L and F may be linked to each other by covalent bonds, wherein the conjugate is in the order of formula (1), with X, L, and F respectively through covalent bonds It may also be a linked conjugate.

In addition, X may be directly linked to F (that is, a is 0 in Formula (1)) or linked via a linker (L).

As one specific example, La, which is one component of the long-lasting conjugate of formula (1), is a linker containing ethylene glycol repeating units, such as polyethylene glycol. These derivatives that are already known and derivatives that can be easily produced at the technical level in the field are also included in the scope of the present invention.

The linker L comprising the ethylene glycol repeating unit may be terminated with a functional group used to prepare the conjugate before it is formed into a conjugate. The long-acting conjugate according to the present invention may have a form in which X and F are linked through the functional group, but is not limited thereto. In the present invention, the linker containing the ethylene glycol repeating unit may include 2 or 3 or more functional groups, and each functional group may be the same or different, but is not limited thereto.

Specifically, the linker may be polyethylene glycol (PEG) represented by the following chemical formula (3), but is not limited thereto:

・・・（３）

... (3)

Here, n=10-2400, n=10-480, or n=50-250, but not limited thereto.
Part of the PEG in said long-acting conjugates includes not only the —(CH ₂ CH ₂ O) _n — structure, but also the oxygen atom intervening between the linking member and this —(CH ₂ CH ₂ O) _n —. may include, but is not limited to.

The polyethylene glycol is a term encompassing all forms of ethylene glycol homopolymer, PEG copolymer, or monomethyl-substituted PEG polymer (mPEG), but is not limited thereto.

As one specific example, the ethylene glycol repeating unit can be represented by [OCH ₂ CH ₂ ]n, where the value of n is a natural number and represents the number of [OCH ₂ CH ₂ ]n sites in the peptide conjugate. The average molecular weight, eg, number average molecular weight, may be determined to be from greater than 0 to about 100 kDa, but is not so limited. As another example, the n value is a natural number and the average molecular weight of the [OCH ₂ CH ₂ ]n site in the peptide conjugate, for example, the number average molecular weight is about 1 to about 100 kDa, about 1 to about 80 kDa, about 1 to about 50 kDa, about 1 to about 30 kDa, about 1 to about 25 kDa, about 1 to about 20 kDa, about 1 to about 15 kDa, about 1 to about 13 kDa, about 1 to about 11 kDa, about 1 to about 10 kDa, about 1 to about 8 kDa , about 1 to about 5 kDa, about 1 to about 3.4 kDa, about 3 to about 30 kDa, about 3 to about 27 kDa, about 3 to about 25 kDa, about 3 to about 22 kDa, about 3 to about 20 kDa, about 3 to about 18 kDa. , about 3 to about 16 kDa, about 3 to about 15 kDa, about 3 to about 13 kDa, about 3 to about 11 kDa, about 3 to about 10 kDa, about 3 to about 8 kDa, about 3 to about 5 kDa, about 3 to about 3.4 kDa. , about 8 to about 30 kDa, about 8 to about 27 kDa, about 8 to about 25 kDa, about 8 to about 22 kDa, about 8 to about 20 kDa, about 8 to about 18 kDa, about 8 to about 16 kDa, about 8 to about 15 kDa, about 8 to about 13 kDa, about 8 to about 11 kDa, about 8 to about 10 kDa, about 9 to about 15 kDa, about 9 to about 14 kDa, about 9 to about 13 kDa, about 9 to about 12 kDa, about 9 to about 11 kDa, about 9. It may be from 5 to about 10.5 kDa, or about 10 kDa, but is not so limited.

As one specific example, both ends of the linker may be bound to the thiol group, amino group, hydroxyl group of the immunoglobulin Fc segment and the thiol group, amino group, azide group, hydroxyl group of the peptide of general formula 1. , but not limited to.

Specifically, the linker has a reactive group that can be bound to immunoglobulin Fc and a peptide of general formula 1 at both ends, specifically, a cysteine thiol group of immunoglobulin Fc fragment; N-terminus, lysine, arginine, glutamine and/or an amino group located at histidine; and/or a thiol group of cysteine of the peptide of general formula 1, bound to the hydroxyl group located at the C-terminus; an amino group of lysine, arginine, glutamine and/or histidine; may include, but are not limited to, reactive groups that may be attached to an azide group; and/or a hydroxyl group.

More specifically, the reactive group of the linker may be one or more selected from the group consisting of an aldehyde group, a maleimide group and a succinimide derivative, but is not limited thereto.

In the above, the aldehyde group can be exemplified by a propionaldehyde group or a butyraldehyde group, but is not limited thereto.

In the above, the succinimide derivatives include succinimidyl carboxymethyl, succinimidyl valeric acid, succinimidyl methylbutanoic acid, succinimidyl methyl propionic acid, succinimidyl butanoic acid, succinimidyl propionic acid, N- Hydroxysuccinimide, hydroxysuccinimidyl or succinimidyl carbonate may be used, but are not limited thereto.

The linker may be linked to the immunoglobulin Fc segment F and the peptide X of the general formula 1 through the reactive group as described above and converted into a linker linker.
Also, the final products produced by reductive alkylation via an aldehyde bond are much more stable than those linked via an amide bond. The aldehyde reactive group selectively reacts with the N-terminus at low pH and can form covalent bonds with lysine residues at high pH, eg, pH 9.0, but is not limited thereto.

The terminal reactive groups of the linkers of the invention may be the same or different from each other. The linker may have an aldehyde group-reactive group at the end, and the linker may have an aldehyde group and a maleimide-reactive group at the end, respectively, or an aldehyde group and a succinimide-reactive group at the end, respectively. Although it may have a group, it is not limited to this.

For example, it can have a maleimide group on one end and an aldehyde, propionaldehyde or butyraldehyde group on the other end. Also, as one example, it can have a succinimidyl group at one end and a propionaldehyde group or a butyraldehyde group at the other end.

When polyethylene glycol having a hydroxy reactive group at the end on the propion side is used as a linker, the hydroxy group can be activated with the various reactive groups by known chemical reactions, or modified reactive groups that are commercially available can be used. can be used to prepare the conjugates of the invention.

In one specific embodiment, the reactive group of the linker may be linked to the cysteine residue of the peptide of general formula 1, more specifically -SH group of cysteine, but is limited to not.

If maleimide-PEG-aldehyde is used, the maleimide group is linked to the -SH group of the peptide of general formula 1 via a thioether bond, and the aldehyde group is reductively alkylated with the _-NH2 group of immunoglobulin Fc. can be connected through, but not limited to, this is an example.

の構造を含んでもよい。 Through such reductive alkylation, the N-terminal amino group of the immunoglobulin Fc segment is linked to the oxygen atom located at one end of PEG through a linker functional group having a structure of -CH ₂ CH ₂ CH ₂ -, and - A structure such as PEG-O-CH ₂ CH ₂ CH ₂ NH-immunoglobulin Fc can be formed, and one end of PEG is linked to the sulfur atom located at the cysteine of the peptide of general formula 1 through a thioether bond. can be formed. The thioether bond mentioned above is

may contain the structure of

However, it is not particularly limited to the example described above, and this corresponds to one example.

Also, in the conjugate, the reactive group of the linker may be linked to --NH ₂ located at the N-terminus of the immunoglobulin Fc segment, but this corresponds to one example.

Also, in the conjugate, the peptide of general formula 1 may be linked to a linker having a reactive group through the C-terminus, but this is an example.

In the present invention, the term "C-terminus" means the carboxy terminus of a peptide, and for the purposes of the present invention, the position at which a linker can be attached. Examples thereof include, but are not limited to, not only the most terminal amino acid residue of the C-terminus, but also any amino acid residue around the C-terminus, specifically the 1st to 20th from the most terminal It may contain, but is not limited to, amino acid residues.

As one specific example, the conjugate of Formula (1) may have a structure of Formula (2) below.

・・・（２）

... (2)

In the chemical formula (2), X is the peptide of general formula 1 described above,
F is the human immunoglobulin Fc segment;
n may be a natural number. The explanation for n is then as above.

As one specific example, the long-acting conjugate of formula (2) is a peptide X of general formula 1 of SEQ ID NO: 46 and a human immunoglobulin Fc segment F covalently linked via an ethylene glycol repeat. wherein X is linked to the succinimide ring of chemical formula (2) and F is linked to the oxypropylene group of chemical formula (2).

In the chemical formula (2), the value of n is adjusted so that the average molecular weight of the [OCH ₂ CH ₂ ]n site in the peptide conjugate, for example, the number average molecular weight is 1 to 100 kDa, 1 to 20 kDa, or 10 kDa. It may be defined, but is not limited to this.

X of the peptide conjugate may be a peptide having activity against glucagon derivatives.

In one embodiment, the site where X is linked to the succinimide ring of formula (2) may be the sulfur atom of the C-terminal cysteine of X.

F is a human immunoglobulin Fc segment, and reference herein to a human immunoglobulin Fc segment includes not only the native sequence obtained from papain digestion of immunoglobulin, but also derivatives, substitutions thereof, e.g. Variants such as sequences differing from the natural type due to deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues, or a combination thereof are also included. It is assumed that said derivatives, substitutions, variants retain the ability to bind to FcRn.

The site linked to the oxypropylene group in F is not particularly limited. In one embodiment of the invention, the portion of F linked to the oxypropylene group may be the N-terminal nitrogen or the nitrogen atom of an internal residue of F (eg, the epsilon nitrogen of lysine). In one specific embodiment of the present invention, the site where F is linked to the oxypropylene group of formula (1) may be the N-terminal proline of F, but is not limited thereto.

The F is a structure in which two polypeptide chains are linked by a disulfide bond, and may be a structure in which one of the two chains is linked only through a nitrogen atom, but is not limited thereto. . The linkage through the nitrogen atom may be linked to the epsilon amino atom of lysine or the N-terminal amino group through reductive amination, but is not limited thereto.

Reductive amination reaction is the reaction of an amine group or amino group of a reactant with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to form an amine, followed by a reduction reaction to form an amine. It means a reaction that forms a bond, and is an organic synthesis reaction that is widely known in the art.

As one specific example, the F may be linked through the nitrogen atom of its N-terminal proline, but is not limited thereto.

Types of the glucagon derivative peptides include those described in International Publication WO2016/108586 and International Publication WO2017/003191, and the entire specifications of the International Publication are included in the present application as reference materials. be In addition, the method for producing the long-acting conjugate of the glucagon derivative peptide is described in WO2017/003191, and it is clear that the entire specification of the international publication is also included in the present application as a reference.

Such glucagon derivatives may have an altered pI relative to native glucagon and exhibit improved physical properties. Also, the glucagon derivative may have improved solubility while having the activity of activating the glucagon receptor, but is not limited thereto.

Also, the glucagon derivative may be non-naturally occurring.

Alternatively, native glucagon can have the following amino acid sequence:

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp- Leu-Met-Asn-Thr (SEQ ID NO: 1)

As used herein, the term "isoelectric point" or "pI" means the pH value at which a molecule such as a polypeptide or peptide loses its overall net charge (0). For polypeptides in which multiple charged functional groups are present, the sum of these charges is zero at the pI. At pH values above the pI, the overall net charge of the polypeptide should be negative, and at pH values below the pI, the overall net charge of the polypeptide should be positive.

The pI was determined by isoelectric focusing on a fixed pH gradient gel composed of polyacrylamide, starch or agarose, or by e.g. the pI/MW tool (http://expasy.org/tools/ pi_tool.html; Gasteiger et al., 2003) to estimate the pI from the amino acid sequence.

In the present invention, the term "altered pi" means that the amino acid sequence of native glucagon is partially substituted with negatively and positively charged amino acid residues in the sequence and differs from the pi of native glucagon, i.e., from this It means having a decreased or increased pI. Peptides with such altered PI can exhibit improved solubility and/or increased stability at neutral pH as glucagon derivatives. However, it is not particularly limited to this.

More specifically, the glucagon derivative may have an altered pI value rather than the pI value of native glucagon (6.8), more specifically less than 6.8, specifically may be a pI value that is 6.7 or less, more specifically 6.5 or less, also specifically greater than 6.8, 7 or more, more specifically 7.5 or more, It is not limited to this, and is included in the scope of the present invention as long as it has a pI value different from that of native glucagon. In particular, if it has a pI value different from that of native glucagon, thereby exhibiting improved solubility at neutral pH compared to native glucagon, and is less likely to be aggregated, it is particularly within the scope of the present invention. included.

More specifically, a pI value of 4 to 6.5 and/or 7 to 9.5, more specifically 7.5 to 9.5, even more specifically 8.0 to 9.3. Although it may have, it is not restricted to this. In this case, it has a higher or lower pI than native glucagon, and thus can exhibit improved solubility and greater stability than native glucagon at neutral pH. However, it is not limited to this.

Specifically, a derivative of natural glucagon is obtained by any one method of substitution, addition, deletion and modification of some amino acids in natural glucagon, or such Variations can be made through a combination of methods. Substitutions of said amino acids generally occur on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues, and amino acids having such similar properties Conservative substitutions can be expected to exhibit the same or similar activity.

Examples of derivatives of glucagon produced by a combination of these methods include activity against the glucagon receptor, which differs from natural glucagon in one or more amino acid sequences and is deaminated at the N-terminal amino acid residue. Examples include, but are not limited to, peptides possessing a catalyzing function, and derivatives of natural glucagon applicable to the present invention can be produced by combining various methods for producing derivatives.

Also, such modifications for the production of derivatives of native glucagon include modifications with L- or D-form amino acids and/or unnatural amino acids, and/or modifications of the native sequence, such as modification of side chain functional groups. Modifications, modifications such as intramolecular covalent bonds, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexane, biotinylation, etc. are all included. Said variations also include any substitution of non-naturally occurring compounds.

Furthermore, the derivatives of natural glucagon include all those with at least one amino acid added to the amino and/or carboxy terminus of natural glucagon.

As the amino acids to be substituted or added, not only the 20 kinds of amino acids normally observed in human proteins but also abnormal or non-naturally occurring amino acids can be used. Commercial sources of unusual amino acids include Sigma-Aldrich, ChemPep, Genzyme pharmaceuticals. Peptides containing these amino acids and standard peptide sequences can be synthesized and purchased from private peptide synthesis companies, such as American peptide company and Bachem in the United States, or Anygen in Korea.

Amino acid derivatives are also available in the same manner, one example being 4-imidazoacetic acid.

Glucagon has a pI of about 7, is insoluble in solutions at physiological pH (pH 4-8), and tends to precipitate at neutral pH. In aqueous solutions of pH 3 or less, glucagon initially dissolves, but precipitates by gel formation within 1 hour. Gelled glucagon is primarily composed of β-sheet fibrils, and such precipitated glucagon is used as an injectable agent because the gel causes it to occlude blood vessels when administered via a needle or intravenously. not suitable for To slow the precipitation process, it is common to use acidic (pH 2-4) formulations through which glucagon can be kept relatively unaggregated for short periods of time. However, since glucagon fibril formation occurs very rapidly at low pH, such acidic dosage forms must be injected immediately after preparation.

In contrast, in the present invention, a glucagon derivative having an extended action profile was developed by altering the pI of natural glucagon by substituting negatively and positively charged amino acid residues. The glucagon derivatives of the present invention are characterized in that they can exhibit improved solubility and/or increased stability at neutral pH by having an altered pI compared to native glucagon.

In one specific embodiment, the glucagon derivative may be a peptide comprising the amino acid sequence of general formula 1 below.

Y-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T- X30 (general formula 1, SEQ ID NO: 46)

In the above formula,
X2 is α-methyl-glutamic acid, Aib (aminoisobutylic acid), D-alanine, glycine, Sar (N-methylglycine), serine or D-serine;
X7 is threonine, valine or cysteine;
X10 is tyrosine or cysteine;
X12 is lysine or cysteine,
X13 is tyrosine or cysteine;
X14 is leucine or cysteine,
X15 is aspartic acid, glutamic acid or cysteine;
X16 is glutamic acid, aspartic acid, serine, α-methylglutamic acid, or cysteine, or is absent;
X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine, or valine, or absent;
X18 is alanine, aspartic acid, glutamic acid, glutamine, arginine, valine, or cysteine, or is absent;
X19 is alanine, arginine, serine, valine, cysteine, or absent;
X20 is lysine, histidine, glutamic acid, glutamine, aspartic acid, arginine, α-methylglutamic acid, or cysteine, or is absent;
X21 is aspartic acid, glutamic acid, leucine, valine, cysteine, or absent;
X23 is isoleucine, valine, arginine, or absent;
X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, α-methyl-glutamic acid, or leucine, or absent;
X27 is isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine, or absent;
X28 is glutamine, lysine, asparagine, arginine, or absent;
X30 may be cysteine or may be absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

More specifically,
In the general formula 1,
X2 is serine or Aib (aminoisobutylic acid);
X7 is threonine, valine or cysteine;
X10 is tyrosine or cysteine;
X12 is lysine or cysteine,
X13 is tyrosine or cysteine;
X14 is leucine or cysteine,
X15 is aspartic acid or cysteine,
X16 is glutamic acid, serine or cysteine;
X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine;
X18 is aspartic acid, glutamic acid, arginine, or cysteine;
X19 is alanine or cysteine,
X20 is glutamine, aspartate, lysine, or cysteine;
X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine;
X23 is isoleucine, valine or arginine;
X24 is valine, arginine, alanine, glutamic acid, lysine, glutamine, or leucine;
X27 is isoleucine, valine, alanine, methionine, glutamine or arginine;
X28 is glutamine, lysine, asparagine or arginine;
X30 may be cysteine or absent. (Except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

For example, the glucagon derivative peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45, specifically, an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45 ( (mandatory), but not limited to.

In addition, even if it is described as "a peptide composed of a specific SEQ ID NO" in the present application, if it has the same or corresponding activity as a peptide composed of the amino acid sequence of the SEQ ID NO, the amino acid sequence of the SEQ ID NO without excluding insignificant sequence additions or naturally occurring mutations before or after , or silent mutations thereof, even if having such sequence additions or mutations It is self-evident that it belongs to the range.

The above content may be applied to other embodiments or other aspects of the present invention, but is not limited thereto.

Specifically, in the general formula 1,
X2 is serine or Aib (aminoisobutylic acid);
X7 is cysteine, threonine, or valine;
X10 is tyrosine or cysteine;
X12 is lysine or cysteine,
X13 is tyrosine or cysteine;
X14 is leucine or cysteine,
X15 is aspartic acid or cysteine,
X16 is glutamic acid, serine or cysteine;
X17 is glutamic acid, lysine, arginine, cysteine, or valine;
X18 is arginine or cysteine,
X19 is alanine or cysteine,
X20 is glutamine or lysine,
X21 is aspartic acid, glutamic acid, valine, or cysteine;
X23 is valine,
X24 is valine or glutamine;
X27 is methionine,
X28 is asparagine or arginine;
X30 may be cysteine or may be absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

For example, the glucagon derivative peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11-17, 19-27, 29, 31, 33, 35-44, specifically, SEQ ID NO: 3, 11-17, 19-27, 29, 31, 33, 35-44 may be (essentially) composed of an amino acid sequence selected from the group consisting of, but not limited to not.

Specifically, in the general formula 1, X2 is Aib (aminoisobutylic acid);
X7 is cysteine, threonine, or valine;
X10 is tyrosine or cysteine;
X12 is lysine,
X13 is tyrosine or cysteine;
X14 is leucine or cysteine,
X15 is aspartic acid or cysteine,
X16 is glutamic acid, serine or cysteine;
X17 is lysine, arginine, cysteine, or valine;
X18 is arginine or cysteine,
X19 is alanine or cysteine,
X20 is glutamine or lysine,
X21 is aspartic acid, glutamic acid, or cysteine;
X23 is valine,
X24 is glutamine;
X27 is methionine,
X28 is asparagine or arginine;
X30 is cysteine or may be absent (except when the amino acid sequence of general formula 1 is identical to SEQ ID NO: 1 and SEQ ID NO: 12).

For example, the glucagon derivative peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, 19-25, 27, 29, 33, 35-38, 40-42, and 44; Specifically, those composed (essentially) of an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 17, 19-25, 27, 29, 33, 35-38, and 40-42, 44 However, it is not limited to this.

Specifically, in the general formula 1, X2 is serine or Aib (aminoisobutylic acid);
X7 is threonine, valine or cysteine;
X10 is tyrosine or cysteine;
X12 is lysine or cysteine,
X13 is tyrosine or cysteine;
X14 is leucine or cysteine,
X15 is aspartic acid or cysteine,
X16 is glutamic acid, serine or cysteine;
X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine;
X18 is aspartic acid, glutamic acid, arginine, or cysteine;
X19 is alanine or cysteine,
X20 is glutamine, aspartic acid, or lysine;
X21 is aspartic acid or glutamic acid,
X23 is valine,
X24 is valine or glutamine;
X27 is isoleucine or methionine,
X28 is asparagine or arginine;
X29 is threonine,
X30 is cysteine or may be absent (except when the amino acid sequence of general formula 1 is identical to SEQ ID NO: 1 and SEQ ID NO: 12).

As an example thereof, the glucagon derivative peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2-13, 15, 17, 20-24, 26-30, and 32-44, specifically the sequence It may be (essentially) composed of an amino acid sequence selected from the group consisting of numbers 2 to 13, 15, 17, 20 to 24, 26 to 30, and 32 to 44, but is not limited thereto. .

Specifically, in the general formula 1, X2 is Aib (aminoisobutylic acid);
X7 is threonine,
X10 is tyrosine,
X12 is lysine,
X13 is tyrosine,
X14 is leucine,
X15 is aspartic acid or cysteine,
X16 is glutamic acid, serine or cysteine;
X17 is lysine or arginine;
X18 is arginine;
X19 is alanine,
X20 is glutamine, cysteine, or lysine;
X21 is aspartic acid, cysteine, valine or glutamic acid;
X23 is valine or arginine;
X24 is glutamine or leucine,
X27 is methionine,
X28 is asparagine or arginine;
X29 is threonine,
X30 may be absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

For example, the glucagon derivative peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 14, 16, 18, 19, 25, and 31, specifically SEQ ID NOS: 14, 16, 18 , 19, 25, and 31 (essentially), but not limited thereto.

More specifically, the glucagon derivative peptide may be a peptide comprising the amino acid sequence of general formula 2 below:

Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-NT- X30 (general formula 2, SEQ ID NO: 47)

In the general formula 2, X7 is threonine, valine or cysteine,
X10 is tyrosine or cysteine;
X12 is lysine or cysteine,
X15 is aspartic acid or cysteine,
X16 is glutamic acid or serine;
X17 is lysine or arginine;
X20 is glutamine or lysine,
X21 is aspartic acid or glutamic acid,
X24 is valine or glutamine;
X30 may be cysteine or may be absent (except when the amino acid sequence of general formula 2 is the same as SEQ ID NO: 1 and SEQ ID NO: 12).

For example, the glucagon derivative peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15 and 36-44, specifically the group consisting of SEQ ID NOs: 13, 15 and 36-44. It may be (essentially) composed of an amino acid sequence selected from, but is not limited to. More specifically, the peptide comprises the amino acid sequence of SEQ ID NO: 20 or 37, or consists (essentially) of the amino acid sequence. However, it is not limited to this.

Specifically, in the general formula 2
X7 is threonine, valine or cysteine;
X10 is tyrosine or cysteine;
X12 is lysine,
X15 is aspartic acid,
X16 is glutamic acid or serine;
X17 is lysine or arginine;
X20 is glutamine or lysine,
X21 is aspartic acid or glutamic acid,
X24 is glutamine;
X30 may be cysteine or may be absent (except when the amino acid sequence of general formula 2 is the same as SEQ ID NO: 1 and SEQ ID NO: 12), but is not particularly limited to this. do not have.

For example, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-38, 40-42, and 44, specifically SEQ ID NOs: 36-38, 40-42, and It may consist of (essentially) an amino acid sequence selected from the group consisting of 44, but is not limited to this.

The glucagon derivatives mentioned above may contain intramolecular bridges (eg, covalent or non-covalent bridges), and in particular may be in a form containing a ring. For example, the glucagon derivative may have a form in which a ring is formed between the 16th and 20th amino acids, but is not particularly limited thereto.

Non-limiting examples of such rings may include lactam bridges (or lactam rings).

In addition, the glucagon derivative includes any one modified to include an amino acid capable of forming a ring at the target position so as to include a ring.

Such a ring may be formed between side chains of amino acids in the glucagon derivative, for example, a form in which a lactam ring is formed between a lysine side chain and a glutamic acid side chain. However, it is not particularly limited to this.

For example, the peptide containing the amino acid sequence of general formula 1 or general formula 2 is X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 of general formula 1 or general formula 2. , the amino acid in each amino acid pair may be substituted with glutamic acid or lysine, but is not limited to this. In the above Xn (n is a natural number), n indicates the amino acid position from the N-terminus of the presented amino acid sequence.

In addition, the peptide containing the amino acid sequence of general formula 1 or general formula 2 is glutamic acid or It may be substituted with lysine, but is not limited to this.

In at least one of the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in the general formula 1 or 2, A ring (eg, lactam ring) may be formed between amino acids, but is not limited to this.

Moreover, in the general formula 1 or 2, X16 may be glutamic acid, X20 may be lysine, and the side chains of X16 and X20 may form a lactam ring, but are not limited thereto.

In addition, the glucagon derivative peptide according to the present invention may have an unmodified N-terminus and/or C-terminus. The peptides of the present invention also include modified forms in which the terminus and/or C-terminus are chemically modified, protected with an organic group, or amino acids are added to the peptide terminus. When the C-terminus is not modified, the terminal of the peptide according to the present invention has a carboxyl group, but is not particularly limited thereto.

In particular, in the case of chemically synthesized peptides, the N- and C-termini are charged, so in order to remove such charges, the N-terminus is acetylated and/or the C-terminus is amidated ( amidation) may be performed, but is not particularly limited to these.

Unless otherwise indicated herein, the detailed description and claims of the "glucagon derivative peptides" according to the invention or "conjugates" in which such peptides are covalently linked to an immunoglobulin Fc segment. The range statement applies to categories that include any of the peptides or conjugates as well as salts of the peptides or conjugates (e.g., pharmaceutically acceptable salts of the peptides) or solvate forms thereof. be done. Therefore, even if only "peptide" or "conjugate" is described in the specification, the description applies equally to the specific salt, the specific solvate thereof, and the specific solvate of the specific salt. be done. Such a salt form may be, for example, a form using any pharmaceutically acceptable salt. The type of salt is not particularly limited. However, it is preferably in a form that is safe and effective for individuals such as mammals, but is not particularly limited to this.

In the present invention, "immunoglobulin Fc segment" means the heavy chain constant region of immunoglobulin, excluding heavy and light chain variable regions. Specifically, the immunoglobulin Fc segment may comprise portions of heavy chain constant region 2 (CH2) and/or heavy chain constant region 3 (CH3), more specifically, hinge region (meaning the whole or part of the hinge region).

The immunoglobulin Fc fragment is a constituent part of the peptide conjugate of Formula (1) of the present invention, and specifically, it can correspond to F in Formula (1). Such immunoglobulin Fc segments may include, but are not limited to, the hinge portion in the heavy chain constant region.

In the present invention, the immunoglobulin Fc segment may contain a specific hinge sequence at the N-terminus.

The term "hinge sequence" of the present invention refers to the site located in the heavy chain that forms dimers of immunoglobulin Fc segments through inter disulfide bonds.

In the present invention, the hinge sequence may be mutated to have only one cysteine residue by deleting a portion of the hinge sequence having the following amino acid sequence, but is not limited thereto: :

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO:48).

The hinge sequence may contain only one cysteine residue by deleting the 8th or 11th cysteine residue in the hinge sequence of SEQ ID NO:48. The hinge sequence of the present invention may consist of 3 to 12 amino acids containing only one cysteine residue, but is not limited thereto. More specifically, the hinge sequence of the invention can have the sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 52). , Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 53), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 54), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 55), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 56), Glu -Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO:57), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO:58), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 59), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 60), Pro-Ser-Cys-Pro (SEQ ID NO: 61), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser- Cys-Pro (SEQ ID NO: 62), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 63), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro ( SEQ ID NO: 64), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 65), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 66), Glu-Ser-Lys -Pro-Ser-Cys-Pro (SEQ ID NO:67), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO:68), Glu-Pro-Ser-Cys (SEQ ID NO:69). SEQ ID NO: 49 (Ser-Cys-Pro) or SEQ ID NO: 50 (Pro-Ser-Cys-Pro), more particularly said hinge sequence is SEQ ID NO: 49 (Ser-Cys-Pro) or SEQ ID NO: 50 (Pro -Ser-Cys-Pro) amino acid sequence, but is not limited to this.

The immunoglobulin Fc segment of the present invention may be in the form of a dimer formed by two immunoglobulin Fc chain molecules due to the presence of a hinge sequence. may be in a form in which one end of is linked to one chain of a dimeric immunoglobulin Fc fragment, but is not limited to this.

The term "N-terminus" of the present invention means the amino terminus of a protein or polypeptide, the extreme amino terminus, or the extreme 1, 2, 3, 4, 5 , 6, 7, 8, 9, or up to 10 or more amino acids. The immunoglobulin Fc segment of the present invention may have a hinge sequence at its N-terminus, but is not limited to this.

In addition, the immunoglobulin Fc segment of the present invention may contain a part or the whole of the heavy chain constant region, excluding only the immunoglobulin heavy and light chain variable regions, as long as the effect is substantially equivalent to or improved from that of the native type. 1 (CH1) and/or light chain constant region 1 (CL1). Alternatively, it may be a region from which a considerably long partial amino acid sequence corresponding to CH2 and/or CH3 has been removed.

For example, the immunoglobulin Fc segments of the present invention may comprise 1) CH1, CH2, CH3 and CH4 domains, 2) CH1 and CH2 domains, 3) CH1 and CH3 domains, 4) CH2 and CH3 domains, 5 ) a combination of one or more domains in the CH1 domain, CH2 domain, CH3 domain and CH4 domain with an immunoglobulin hinge region (or part of the hinge region); 6) each domain of the heavy chain constant region and the light chain constant region; It may be a dimer of constant regions. However, it is not limited to this.

In one embodiment, the immunoglobulin Fc segment may be in a dimeric form, wherein one molecule of the peptide of general formula 1 is covalently bound to one Fc region of the dimeric form. at which time the immunoglobulin Fc and the peptide of general formula 1 may be linked to each other by a linker containing ethylene glycol repeating units. On the other hand, it is also possible for two molecules of the peptide of general formula 1 to be symmetrically bound to one Fc region in dimeric form. The immunoglobulin Fc and the peptide of general formula 1 may then be linked to each other by a linker containing ethylene glycol repeating units. However, it is not limited to the above examples.

In addition, the immunoglobulin Fc segment of the present invention includes not only naturally occurring amino acid sequences, but also sequence derivatives thereof. An amino acid sequence derivative means having a sequence that differs by deletion, insertion, non-conservative or conservative substitution, or a combination thereof of one or more amino acid residues in the native amino acid sequence.

For example, in the case of IgG Fc, amino acid residues 214-238, 297-299, 318-322 or 327-331, which are known to be important for binding, can be used as suitable sites for modification. .

In addition, sites that can form disulfide bonds can be removed, some amino acids at the N-terminus can be removed from the native Fc, or a methionine residue can be added to the N-terminus of the native Fc. Various types of derivatives are possible. Also, the complement binding site, eg, the C1q binding site, may be removed to abolish effector function, and the ADCC (antibody dependent cell mediated cytotoxicity) site may be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc segments are disclosed in International Patent Publication No. WO97/34631, International Patent Publication No. 96/32478, and the like.

Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Exchanges between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. optionally phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation and amidation ) and the like.

The Fc derivative may exhibit biological activity equivalent to that of the Fc segment of the present invention and may have increased structural stability against heat, pH, etc. of the Fc region.

Such Fc segments may also be obtained from native forms isolated from in vivo animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs, transformed animal cells or It may be a recombinant form obtained from a microorganism or a derivative thereof. Here, the method of obtaining from the natural type may be a method of obtaining whole immunoglobulin by isolating it from a human or animal body and treating it with a proteolytic enzyme. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated from this by using size-exclusion chromatography or the like. In a more specific embodiment, the human-derived Fc segment is a microbial recombinant immunoglobulin Fc segment.

In addition, the immunoglobulin Fc segment may be in the form of native sugar chains, sugar chains increased compared to the native type, sugar chains decreased compared to the native type, or sugar chains removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase, decrease, or remove immunoglobulin Fc sugar chains. Here, the immunoglobulin Fc fragment in which sugar chains are removed from Fc has significantly reduced binding power with complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated. , does not induce unwanted immune responses in vivo. In this respect, it can be said that a deglycosylated or non-glycosylated immunoglobulin Fc segment is more suitable for its original purpose as a drug carrier.

In the present invention, "deglycosylation" refers to an Fc segment from which sugars have been removed by an enzyme, and non-glycosylation (Aglycosylation) is a prokaryotic animal, and in a more specific embodiment, produced in E. coli. Denotes the non-glycosylated Fc segment.

On the other hand, the immunoglobulin Fc segment may be of human or animal origin such as bovine, goat, porcine, mouse, rabbit, hamster, rat, guinea pig, and in a more specific embodiment is of human origin.

The immunoglobulin Fc segment may also be derived from IgG, IgA, IgD, IgE, IgM, a combination thereof, or a hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which are most abundant in human blood, and in a more specific embodiment, from IgG, which has been shown to improve the half-life of ligand binding proteins. In an even more specific embodiment said immunoglobulin Fc segment is an IgG4 Fc segment, and in a most specific embodiment said immunoglobulin Fc segment is a non-glycosylated Fc segment derived from human IgG4, wherein It is not limited.

Also, in one specific embodiment, the immunoglobulin Fc segment is a fragment of human IgG4 Fc, through a disulfide bond (inter-chain form) between the third amino acid cysteine of each monomer. It may also be in the form of a homodimer in which two monomers are linked, in which case each monomer of the homodimer independently has an internal It has/can have a disulfide bond and an internal disulfide bond between cysteines at positions 141 and 199, ie, two internal disulfide bonds (intra-chain form). The number of amino acids in each monomer may consist of 221 amino acids, and the amino acids forming the homodimer may consist of a total of 442 amino acids, but is not limited thereto. Specifically, the immunoglobulin Fc segment consists of two monomers having the amino acid sequence of SEQ ID NO: 51 (composed of 221 amino acids) with a disulfide bond between the cysteine at the third amino acid of each monomer. and the homodimer monomers independently form internal disulfide bonds between cysteines 35 and 95 and internal disulfide bonds between cysteines 141 and 199. , but is not limited to this.

F in the chemical formula (1) may contain a monomer having the amino acid sequence of SEQ ID NO: 51, and the F is a homodimer of the monomer having the amino acid sequence of SEQ ID NO: 51, can be, but is not limited to.

F in the chemical formula (1) may contain a monomer having the amino acid sequence of SEQ ID NO: 51, and the F is a homodimer of the monomer having the amino acid sequence of SEQ ID NO: 51, can be, but is not limited to.

As an example, but not limited to, the immunoglobulin Fc segment may be a homodimer comprising the amino acid sequence of SEQ ID NO: 70 (consisting of 442 amino acids).

On the other hand, in the present invention, "combination" means that when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin Fc segment of the same origin binds to a single-chain polypeptide of a different origin. means to form Thus, dimers or multimers can be produced from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc fragments.

As used herein, the term "hybrid" refers to the presence of sequences corresponding to two or more immunoglobulin Fc fragments of different origins within a single immunoglobulin constant region. Various forms of hybrids are possible with the present invention. That is, it is possible to hybridize a domain consisting of 1 to 4 domains from the group consisting of CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, including the hinge. good.

On the other hand, IgG can also be divided into IgG1, IgG2, IgG3 and IgG4 subclasses, and combinations or hybridizations thereof are also possible in the present invention. Specifically, the IgG2 and IgG4 subclasses, most specifically the Fc segment of IgG4, which has little effector function such as complement dependent cytotoxicity (CDC).

The liquid formulation is used for prevention or treatment of congenital hyperinsulinism, hypoglycemia, or metabolic syndrome.

In the present invention, the term "prevention" means that administration of the glucagon derivative, a conjugate containing the same, or a composition suppresses or suppresses the onset of a target disease, such as congenital hyperinsulinism, hypoglycemia, or metabolic syndrome. "Treatment" refers to any action that delays the treatment of a disease of interest, such as congenital hyperinsulinism, hypoglycemia, or metabolic syndrome, by administration of the glucagon derivative, conjugate comprising the same, or composition. means any action that improves or benefits the symptoms of

In the present invention, the term "administration" means introducing a given substance into a patient by any appropriate method, and the route of administration of the composition is not particularly limited to this. may be administered via any common route that can reach the target of the drug, such as intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary administration, rectal administration, and the like.

In the present invention, the term "hypoglycemia" refers to a condition in which the blood sugar level is lower than that of a normal person. Usually, it refers to when the blood sugar is 50 mg/dl or less, but it is not particularly limited to this. A common cause of hypoglycemia is when people taking oral hypoglycemic agents or insulin eat and drink less than they normally would, or when they are overactive or exercising. If you also have alcohol or some drugs that lower blood sugar, serious physical illness, deficiencies of hormones such as corticosteroids or glucagon, insulin-producing pancreatic tumors, autoimmune disorders against insulin, gastrectomy Hypoglycemia can also occur in patients, hereditary disorders of carbohydrate-metabolizing enzymes, and the like.

In the present invention, the hypoglycemia includes both acute hypoglycemia and chronic hypoglycemia.

Symptoms of hypoglycemia include lethargy and shivering, pallor, cold sweats, dizziness, agitation, anxiety, palpitations, hunger, headaches, and fatigue. Prolonged hypoglycemia can lead to convulsions and seizures, which can lead to shock and loss of consciousness.

More specifically, said hypoglycemia can be induced by persistent hyperinsulinism due to genetic defects. Causes of hyperinsulinism due to genetic defects include mutation of the SUR or Kir6.2 gene on the 11p15.1 chromosome, GK (glucokinase) gene mutation on the 7p15-p13 chromosome that increases GK activity, and GDH (Glutamate dehydrogenase) gene mutation activates GDH, which is known to increase ATP in beta pancreatic islet cells.

On the other hand, congenital hyperinsulinism is one of the causative diseases that cause severe and persistent hypoglycemia in neonates and children. It can be induced by a transient increase in insulin secretion in low-birth-weight infants or infants of diabetic mothers, abnormal function of pancreatic cells due to genetic mutations, and the like. It is known that glucagon is used for treatment of such congenital hyperinsulinism.

In addition, the glucagon derivative of the present invention or a conjugate containing the same can prevent weight gain, promote weight loss, reduce overweight, and treat obesity including morbid obesity (e.g., appetite, eating, by regulating food intake, calorie intake and/or energy expenditure), as well as related diseases and health including but not limited to obesity-related inflammation, obesity-related gallbladder disease and obesity-induced sleep apnea. Used as a drug to treat conditions. The glucagon derivatives of the present invention or conjugates containing the same are also useful for metabolic syndromes other than obesity, namely impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, hypertension, nonalcoholic steatohepatitis (NASH). , arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease (coronary heart disease), or related diseases such as stroke. However, the effects of glucagon derivatives or conjugates thereof according to the present invention in these disease states may be mediated in whole or in part through the aforementioned weight-related effects, or may be independent of this.

Yet another aspect embodying the present invention provides a method for producing the liquid formulation.

The liquid formulation and the components constituting it are as described above.

Specifically, the production method includes i) a long-acting conjugate of a glucagon derivative peptide in which the glucagon derivative peptide and an immunoglobulin Fc segment are linked to each other, and (ii) (a) a buffer substance and (b) a sugar alcohol, A step of mixing together stabilizing agents including sugars or combinations thereof may also be included.

The stabilizer may further comprise one or more components selected from the group consisting of sugars or sugar alcohols, nonionic surfactants and amino acids, more specifically sugars or sugar alcohols, amino acids , or both. For example, the stabilizing agents may include buffer substances, sugars or sugar alcohols, and amino acids. However, it is not limited to this.

The long-acting conjugates, buffer substances, sugars, sugar alcohols or combinations thereof, nonionic surfactants, amino acids, and albumin-free stabilizing agents are as described above.

Another aspect embodying the present invention provides use of the liquid formulation in the manufacture of a medicament for the prevention or treatment of congenital hyperinsulinism, hypoglycemia, or metabolic syndrome.

Another aspect embodying the present invention provides the use of the liquid formulation for use in preventing or treating congenital hyperinsulinism, hypoglycemia, or metabolic syndrome.
Another aspect embodying the present invention provides a method of preventing or treating congenital hyperinsulinism, hypoglycemia, or metabolic syndrome, comprising administering the liquid formulation to an individual in need thereof.

The liquid formulation, congenital hyperinsulinism, hypoglycemia, and metabolic syndrome are as described above.

The above-mentioned individual is an individual who needs administration of the formulation of the present invention, and includes without limitation individuals who can be treated with the liquid formulation of the present invention, and specifically includes mammals including humans, rats, livestock, and the like.

A therapeutic method of the invention may comprise administering a pharmaceutically effective amount of a liquid formulation. A suitable total daily dose may be administered in single or divided doses as determined by the treating physician within the scope of sound medical judgment. However, for the purposes of this invention, a specific therapeutically effective amount for a particular patient will depend on the specific composition, including the type and extent of response sought to be achieved, and whether other formulations are optionally used. Patient age, body weight, general health, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used with or concurrently with the specific composition, and various other factors. It is preferred to apply differently by analogous factors well known in the pharmaceutical field.

The present invention will now be described in more detail with reference to the following examples. However, the following examples are merely for the purpose of illustrating the present invention, and the scope of the present invention is not limited to these.

Production Example: Production of long-acting conjugate of glucagon derivative peptide The long- acting conjugate of glucagon derivative peptide was produced by the following method.
Maleimido-PEG-aldehyde (Japan NOF) was reacted with the cysteine-containing derivative in the aforementioned glucagon derivative peptide, and the cysteine residue of this glucagon derivative peptide was pegylated at the maleimide-side terminal of maleimide-PEG-aldehyde. Specifically, the glucagon derivative peptide of SEQ ID NO: 37 and maleimide-PEG-aldehyde were reacted at a molar ratio of 1:1-5 at a protein concentration of 3-10 mg/ml at a low temperature for 1-3 hours. At that time, the reaction was carried out in a 50 mM Tris buffer (pH 7.5) with 20-60% isopropanol added. After the reaction was completed, the reaction mixture was applied to SP Sepharose HP (GE Healthcare, USA) to purify the cysteine-monopegylated glucagon derivative.

Immunoglobulin Fc segment, an immunoglobulin Fc segment (49.8 kDa, homodimer in which the two chains of SEQ ID NO: 51 are linked by disulfide bonds) having a hinge region of Pro-Ser-Cys-Pro sequence at the N-terminus using the method described in International Publication WO2007/021129.

Next, the purified monopegylated glucagon derivative peptide and the immunoglobulin Fc fragment are reacted at a molar ratio of 1:2-10 at a protein concentration of 10-50 mg/mL at 4-8° C. for 12-18 hours. let me The reaction was carried out in an environment in which 100 mM potassium phosphate buffer (pH 6.0) was added with 10 to 50 mM sodium cyanoborohydride and 10 to 20% isopropanol as reducing agents. After the reaction was completed, the reaction solution was applied to a Butyl Sepharose FF purification column (GE healthcare, USA) and a Source ISO purification column (GE healthcare, USA), and polyethylene glycol was added to the aldehyde side of the monopegylated glucagon derivative peptide. Long-acting conjugates of glucagon derivative peptides were purified whose ends were linked to the N-terminal proline nitrogen of one chain in the two chains of the immunoglobulin Fc homodimer.

The purity analyzed by reverse phase chromatography, size exclusion chromatography and ion exchange chromatography after preparation was 95% or more.

Here, the conjugate in which the glucagon derivative peptide and the immunoglobulin Fc segment were linked via PEG was named "long-acting conjugate of glucagon derivative peptide".

Example 1: Stability evaluation of long-lasting conjugates of glucagon derivative peptides according to pH and type of sugar or sugar alcohol Variety based on liquid formulations consisting of polysorbate 20 as a buffer substance, a surfactant, a sugar or sugar alcohol, and methionine We compared the stability of long-acting conjugates of glucagon derivative peptides (hereinafter referred to as "glucagon derivatives") under different pH conditions and stabilizing agents. As a comparative example, a formulation with a pH of 4.5 was used.

The long-acting conjugate of the glucagon derivative peptide obtained in the above Preparation Example was prepared into a liquid preparation having the composition shown in Table 1 below (the concentration of the long-acting conjugate was 187.09 nmol/mL) and stored at 25°C for 6 weeks. Later, the ion exchange chromatography method (Ion Exchange High PERFORMANCE LIQUID CHROMATOGRAPLAPHY, IE -HPLC), and the reverse phase chromatography method (Reverse Phase -High PERFORMANN) The stability was analyzed using CE LIQUID CHROMATOGRAPLC).

IE-HPLC (%) and RP-HPLC (%) in Table 2 are percentage values of the ratio (Area%/Start Area%) obtained by dividing the area percentage value at the time of measurement by the initial area percentage value of the storage test, FIG. 4 shows the residual rate of the long-acting glucagon derivative conjugate from the initial concentration (187.09 nmol/mL concentration).

As seen from the above results, it was confirmed that formulations (#1, #4, #7) having a composition of sodium citrate, pH 5.0 exhibited stability.

In addition, the sugars or sugar alcohols mannitol, sorbitol, and sucrose included at 5%, 5%, and 8%, respectively, are included to increase the storage stability of the formulation having a composition of sodium citrate, pH 5.0. , showed similar stability. In the case of the pH 4.5 composition of Comparative Examples 1, 2 and 3, it was confirmed that precipitation occurred in 6 weeks.

Example 2: Stability evaluation of long-acting conjugates of glucagon derivatives depending on the type of buffering substance, pH and type of sugar or sugar alcohol. We compared the stability of long-acting conjugates of glucagon derivatives according to the type and pH. Among them, the stability of the long-acting conjugates of glucagon derivatives added with mannitol, sorbitol and sucrose confirmed in Example 1 was compared. At that time, the concentrations of mannitol, sucrose and sorbitol were considered the maximum acceptable ranges recommended by the commercial dosage forms and licensing agencies.

The long-acting conjugate of the glucagon derivative peptide obtained in the above Preparation Example was prepared into a liquid preparation having the composition shown in Table 3 below (the concentration of the long-acting conjugate was 187.09 nmol/mL) and stored at 25°C for 7 weeks. It was later analyzed using ion-exchange chromatography and reversed-phase chromatography.
IE-HPLC (%) and RP-HPLC (%) in Table 4 are percentage values of the ratio obtained by dividing the area percentage value at the time of measurement by the initial area percentage value of the storage test (Area%/Start Area%), FIG. 4 shows the residual rate of the long-acting glucagon derivative conjugate from the initial concentration (187.09 nmol/mL concentration).

As can be seen from the above results, formulations (#3, #9, #15) with a composition of sodium acetate, pH 5.0 and formulations (#5, #11, #17) with a composition of histidine, pH 5.5 ) showed high stability at 25° C. for 7 weeks. However, it could be confirmed that the preparations (#2, #8, #14) having a composition of pH 4.5 caused precipitation after 7 weeks.

Similar stabilities were shown when the sugars or sugar alcohols mannitol, sorbitol, and sucrose were included at 5%, 5%, and 8%, respectively, to increase the storage stability of the long-acting conjugates of glucagon derivatives. rice field.

Example 3: Evaluation of the stability of long-acting conjugates of glucagon derivatives according to the type of nonionic surfactant to compare the stability of long-acting conjugates of glucagon derivatives with different types of nonionic surfactants. At that time, the concentrations of polysorbate 20, polysorbate 80 and poloxamer 188 as non-ionic surfactants were taken into account in the commercial dosage form.

The long-acting conjugate of the glucagon derivative peptide obtained in the above Preparation Example was prepared into a liquid preparation having the composition shown in Table 5 below (concentration of the long-acting conjugate was 187.09 nmol/mL) and stored at 25°C for 4 weeks. It was later analyzed using ion-exchange chromatography and reversed-phase chromatography.
IE-HPLC (%) and RP-HPLC (%) in Table 6 are percentage values of the ratio (Area%/Start Area%) obtained by dividing the area percentage value at the time of measurement by the initial area percentage value of the storage test, FIG. 4 shows the residual rate of the long-acting glucagon derivative conjugate from the initial concentration (187.09 nmol/mL concentration).

As seen from the above results, it was confirmed that formulations containing polysorbate 20, polysorbate 80 and poloxamer 188 as nonionic surfactants exhibited stability.

Example 4: Stability Evaluation of Long-acting Conjugates of Glucagon Derivatives with or without Nonionic Surfactants and Amino Acids The stability of long-acting conjugates of glucagon derivatives was compared.

The long-acting conjugate of the glucagon derivative peptide obtained in the above Preparation Example was prepared into a liquid preparation having the composition shown in Table 7 below (the concentration of the long-acting conjugate was 187.09 nmol/mL) and stored at 25°C for 4 weeks. It was later analyzed using ion-exchange chromatography and reversed-phase chromatography.

IE-HPLC (%) and RP-HPLC (%) in Table 8 are percentage values of the ratio obtained by dividing the area percentage value at the time of measurement by the initial area percentage value of the storage test (Area%/Start Area%), FIG. 4 shows the residual rate of the long-acting glucagon derivative conjugate from the initial concentration (187.09 nmol/mL concentration).

As seen from the above results, it was confirmed that formulations (#1 and #2) containing amino acids exhibited stability. In addition, it was confirmed that formulations containing nonionic surfactants and formulations without nonionic surfactants showed similar stability to each other.

Example 5: Evaluation of stability of long-acting conjugates of glucagon derivatives depending on pH, sugar concentration and type of amino acid ), the stability of the long-acting conjugate of the glucagon derivative was evaluated based on the pH range in which protein foreign matter or precipitation does not occur, the concentration of sucrose as a sugar, and the types of other amino acids other than methionine.

The long-acting conjugate of the glucagon derivative peptide obtained in the above Preparation Example was prepared into a liquid preparation having the composition shown in Table 9 below (concentration of the long-acting conjugate was 187.09 nmol/mL) and stored at 25°C for 7 weeks. It was later analyzed using ion-exchange chromatography and reversed-phase chromatography.

IE-HPLC (%) and RP-HPLC (%) in Table 10 are percentage values of the ratio (Area%/Start Area%) obtained by dividing the area percentage value at the time of measurement by the storage test initial area percentage value, FIG. 4 shows the residual rate of the long-acting glucagon derivative conjugate from the initial concentration (187.09 nmol/mL concentration).

As can be seen from the above results, when the pH range of the formulation with sodium acetate composition as the buffer solution is pH 4.6 to pH 5.0, the formulation with pH 5.0 composition (#3) is more stable than other pH compositions. It showed excellent stability. Also, in the case of formulation (#1) having a composition of pH 4.6, small particles were generated when stored at 25° C. for 2 weeks, and the number of small particles increased when stored for 5 weeks. As a result of confirming the stability of the glucagon derivative long-acting conjugate depending on the concentration of sucrose, when the sucrose is contained at 0% (#4) to 15% (#8), the higher the concentration of sucrose, the higher the stability tends to be. showed that.

In addition, as a result of confirming the stability of long-acting conjugates of glucagon derivatives according to the type of amino acid, the stability was confirmed in preparations containing arginine (#9), histidine (#10) and lysine (#11).

In other words, these results suggest that the long-acting conjugates of glucagon derivatives are stable in the compositions of the liquid preparations of the present invention in various pH ranges, sugar concentrations, and types of amino acids. .

From the above description, it should be understood by a person skilled in the art to which the present invention pertains that the present invention can be embodied in other specific forms without changing its technical spirit or essential features. deaf. In this regard, it should be understood that the embodiments described above are illustrative only and not limiting. The scope of the present invention should be construed as including any changes or modifications derived from the meaning and scope of the following claims and equivalent concepts thereof, rather than the above detailed description. is.

Claims (31)

A liquid formulation of a long-acting conjugate, wherein the liquid formulation comprises 18-936 nmol/mL of the long-acting conjugate of the following chemical formula (1);
1.0-20% (w/v) sugar alcohols, sugars, or combinations thereof; Liquid formulation:

In the chemical formula (1), X is a glucagon derivative peptide,
L is a linker,
a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
F is the immunoglobulin Fc segment;
- indicates a covalent bond:
[General formula 2]
Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-NT- X30 (general formula 2, SEQ ID NO: 47)
In the general formula 2,
X7 is threonine (T), valine (V) or cysteine (C);
X10 is tyrosine (Y) or cysteine (C);
X12 is lysine (K) or cysteine (C);
X15 is aspartic acid (D) or cysteine (C);
X16 is glutamic acid (E) or serine (S);
X17 is lysine (K) or arginine (R);
X20 is glutamine (Q) or lysine (K);
X21 is aspartic acid (D) or glutamic acid (E);
X24 is valine (V) or glutamine (Q);
X30 is cysteine (C) or absent (except when the amino acid sequence of general formula 2 is the same as SEQ ID NO: 1 and SEQ ID NO: 12). The liquid formulation according to claim 1, wherein the peptide is any one amino acid sequence selected from SEQ ID NOs: 2-11 and 13-45. The liquid formulation according to claim 1, wherein the peptide is any one amino acid sequence selected from SEQ ID NOs: 13, 15 and 36-44. 2. The liquid formulation of claim 1, wherein the peptide is SEQ ID NO:37. 2. The liquid formulation according to claim 1, wherein X is amidated at its C-terminus. 2. The liquid formulation according to claim 1, wherein X is linked through the sulfur atom of cysteine. 2. The liquid formulation of claim 1, wherein said immunoglobulin Fc segment is derived from IgG4. 2. The liquid preparation according to claim 1, wherein F is a structure in which two polypeptide chains are linked by a disulfide bond, and are linked only through the nitrogen atom of one of said two chains. The liquid formulation according to any one of claims 1 to 8, wherein F is a homodimer of monomers of the amino acid sequence of SEQ ID NO:51. 9. The liquid formulation according to claim 8, wherein F is linked through the nitrogen atom of its N-terminal proline. 2. The liquid formulation of claim 1, wherein said immunoglobulin Fc segment and X are non-glycosylated. 2. The liquid formulation according to claim 1, wherein said L is polyethylene glycol. The liquid formulation according to claim 1, wherein the chemical formula weight of the ethylene glycol repeating unit site within L is in the range of 1 to 100 kDa. 2. The liquid formulation according to claim 1, wherein the buffer substance is selected from the group consisting of citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts and combinations thereof. 15. The liquid formulation according to claim 14, wherein the buffer substance is acetic acid and its salts. The liquid formulation according to claim 1, wherein the liquid formulation has a pH of 4.8 to 6.5. The liquid formulation according to claim 1, wherein the liquid formulation has a pH of 4.8 to 6.0. The liquid formulation according to claim 17, wherein the liquid formulation has a pH of 4.8-5.5. The liquid formulation according to claim 1, wherein the concentration of said buffer substance is 5-100 mM for maintaining the pH of the liquid formulation in the range of 4.8-6.5. 2. The liquid formulation of Claim 1, wherein the sugar is glucose, fructose, galactose, lactose, maltose, sucrose, or a combination thereof. 21. A liquid formulation according to claim 20, wherein said sugar is sucrose. 21. The liquid formulation of claim 20, wherein said sugar is present in a concentration of 3-15% (w/v). 2. The liquid preparation according to claim 1, wherein the sugar alcohol is one or more selected from the group consisting of mannitol and sorbitol. 2. The liquid formulation according to claim 1, wherein said liquid formulation further comprises one or more ingredients selected from the group consisting of nonionic surfactants and amino acids. 25. The liquid formulation of claim 24, wherein the nonionic surfactant is at a concentration of 0.01-0.1% (w/v) within the liquid formulation. 25. The liquid formulation of Claim 24, wherein the nonionic surfactant is a poloxamer, polysorbate, or a combination thereof. 27. The liquid formulation of claim 26, wherein the nonionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 and combinations thereof. 25. The liquid formulation of Claim 24, wherein said amino acids further comprise a stabilizer selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine and combinations thereof. 2. The liquid formulation according to claim 1, wherein the liquid formulation does not contain a tonicity agent. The liquid formulation contains 90-562 nmol/mL of the peptide conjugate of formula (1);
5 to 25 mM buffer selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts and combinations thereof so that the pH of the liquid preparation is 4.8 to 5.5 material;
1-20% (w/v) sugar alcohols, sugars, or combinations thereof; and 0.01-0.1% (w/v) nonionic surfactants selected from poloxamers, polysorbates, or combinations thereof. and 0.01-1 mg/mL of a stabilizer selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine and combinations thereof. Liquid formulation as described. The liquid formulation contains 90-562 nmol/mL of the peptide conjugate of formula (1);
5 to 25 mM buffer selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts and combinations thereof so that the pH of the liquid preparation is 4.8 to 5.5 material;
4-10% (w/v) sugars; and 0.01-0.1% (w/v) nonionic surfactants selected from poloxamers, polysorbates or combinations thereof; and methionine, arginine, histidine. , glycine, cysteine, lysine and combinations thereof.

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